T cell-antigen coupler with various construct optimizations

ABSTRACT

A trifunctional molecule is provided, comprising (i) a target-specific ligand, (ii) a ligand that binds a protein associated with a TCR complex, and (iii) a T cell receptor signaling domain polypeptide. Variants of the molecule are provided, including variants that exhibit optimized surface expression, transduction efficiency, and effector functionality. Variations include, for example, different ligands that bind CD3 epsilon (e.g., OKT3, L2K, F6A, UCHT1 and humanized UCHT1), different signaling domains, and different linkers between domains.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/826,053, filed on Mar. 20, 2020, which is a continuation of U.S.application Ser. No. 16/442,274, filed on Jun. 14, 2019, now issued asU.S. Pat. No. 10,640,562 on May 5, 2020, which claims the benefit ofU.S. Provisional Application No. 62/699,173, filed on Jul. 17, 2018,U.S. Provisional Application No. 62/703,037, filed on Jul. 25, 2018,U.S. Provisional Application No. 62/773,120, filed on Nov. 29, 2018,U.S. Provisional Application No. 62/826,853, filed on Mar. 29, 2019,U.S. Provisional Application No. 62/828,879, filed on Apr. 3, 2019, andU.S. Provisional Application No. 62/839,235, filed on Apr. 26, 2019,each of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 17, 2020, isnamed “55247704303_SL.txt” and is 123,321 bytes in size.

SUMMARY

Disclosed herein, in certain embodiments, are nucleic acid sequencesencoding a CD19 Trifunctional T cell-antigen coupler (CD19-TAC). In someembodiments, the nucleic acid sequence encoding a CD19 Trifunctional Tcell-antigen coupler (CD19-TAC) comprises: (a) a first polynucleotideencoding a ligand that selectively binds a CD19 antigen. In someembodiments, the nucleic acid sequence encoding a CD19 Trifunctional Tcell-antigen coupler (CD19-TAC) comprises: (b) a second polynucleotideencoding a UCHT1 ligand that binds CD3. In some embodiments, the nucleicacid sequence encoding a CD19 Trifunctional T cell-antigen coupler(CD19-TAC) comprise: (c) a third polynucleotide encoding a TCR signalingdomain polypeptide comprising a cytosolic domain and a transmembranedomain. In some embodiments, the components encoded by the first,second, and/or third polynucleotides are connected in any suitablemanner, such as in any suitable order and/or comprising any suitablelinker(s). In some embodiments, the components encoded by (a),components encoded by (b), and components encoded by (c) are fuseddirectly to each other, or joined by at least one linker. In someembodiments, the ligand that selectively binds the CD19 antigen is asingle chain variable fragment (scFv). In some embodiments, the ligandthat selectively binds the CD19 antigen comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identitywith SEQ ID NO: 36. In some embodiments, the UCHT1 ligand is a singlechain antibody. In some embodiments, the UCHT1 ligand comprises a Y182Tmutation (SEQ ID NO: 72). In some embodiments, the UCHT1 ligand is ahumanized variant of UCHT1 (huUCHT1) ligand (SEQ ID NO: 44). In someembodiments, the UCHT1 ligand is a humanized variant of UCHT1 comprisinga Y177T mutation (huUCHT1 (Y177T)) (SEQ ID NO: 46). In some embodiments,the UCHT1 ligand comprises an amino acid sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 14,SEQ ID NO: 72, SEQ ID NO: 44, or SEQ ID NO: 46. In some embodiments, thecytosolic domain is a CD4 cytosolic domain and the transmembrane domainis a CD4 transmembrane domain. In some embodiments, the thirdpolynucleotide encodes a polypeptide comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identitywith SEQ ID NO: 18. In some embodiments, the component encoded by (a)and the component encoded by (c) are fused to the component encoded by(b). In some embodiments, the component encoded by (b) and the componentencoded by (c) are fused to the component encoded by (a). In someembodiments, at least one linker joins the component encoded by (a) tothe component encoded by (b). In some embodiments, the at least onelinker is a G₄S flexible linker (SEQ ID NO: 73), a large protein domain,a long helix structure, or a short helix structure. In some embodiments,the at least one linker comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity with SEQ IDNO: 12 (G₄S flexible linker (“G₄S” disclosed as SEQ ID NO: 73)), SEQ IDNO: 32 (large protein domain), SEQ ID NO: 30 (long helix structure), orSEQ ID NO: 28 (short helix structure). In some embodiments, the CD3 isof a TCR complex on a cell expressing the second polynucleotide. In someembodiments, the binding of the CD3 induces activation of a cellexpressing the second polynucleotide. In some embodiments, the CD19-TACcomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity with SEQ ID NO: 63. In someembodiments, the CD19-TAC comprises an amino acid sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withSEQ ID NO: 64. In some embodiments, the nucleic acid sequence does notencode a co-stimulatory domain. In some embodiments, the nucleic acidsequence does not encode an activation domain.

Disclosed herein, in certain embodiments, are vector constructscomprising: (a) a nucleic acid sequence disclosed herein (e.g., anucleic acid sequence encoding a CD19-TAC); and (b) a promoterfunctional in a mammalian cell.

Disclosed herein, in certain embodiments, are T cells comprising anucleic acid sequence disclosed herein (e.g., a nucleic acid sequenceencoding a CD19-TAC).

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising the T cell disclosed herein, and apharmaceutically acceptable excipient.

Disclosed herein, in certain embodiments, are methods of treating cancerexpressing CD19 in an individual in need thereof, comprisingadministering to the individual a pharmaceutical composition disclosedherein. (e.g., a pharmaceutical composition comprising a T cellcomprising any nucleic acid sequence described herein, such as anynucleic acid sequence or sequences described herein as encoding a CD19Trifunctional T cell-antigen coupler (CD19-TAC)). In some embodiments,the cancer is a B cell malignancy. In some embodiments, the cancer is Bcell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), or Non-Hodgkins Lymphoma. In some embodiments, thepharmaceutical composition is administered transarterially,subcutaneously, intradermally, intratumorally, intranodally,intrameduliary, intramuscularly, intravenously or intraperitoneally.

Disclosed herein, in certain embodiments, are nucleic acid sequencesencoding a Trifunctional T cell-antigen coupler (Tri-TAC) comprising:(a) a first polynucleotide encoding a target-specific ligand; (b) asecond polynucleotide encoding a ligand that binds a protein associatedwith a TCR complex; and (c) a third polynucleotide encoding a T cellreceptor signaling domain polypeptide; wherein the ligand that binds theprotein associated with the TCR complex is selected from OKT3, F6A orL2K. In some embodiments, component encoded by (a), component encoded by(b), and component encoded by (c) are fused directly to each other, orjoined by at least one linker. In some embodiments, the componentencoded by (a) and the component encoded by (b) are directly fused andjoined to the component encoded by (c) by a linker. In some embodiments,the component encoded by (b) and the component encoded by (c) aredirectly fused and joined to the component encoded by (a) by a linker.In some embodiments, the at least one linker is a G₄S flexible linker(SEQ ID NO: 73), a large protein domain, a long helix structure, or ashort helix structure. In some embodiments, the at least one linker hasan amino acid sequence having at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with SEQ ID NO: 12 (G₄S flexible linker (“G₄S”disclosed as SEQ ID NO: 73)), SEQ ID NO: 32 (large protein domain), SEQID NO: 30 (long helix structure), or SEQ ID NO: 28 (short helixstructure). In some embodiments, the ligand that binds the proteinassociated with the TCR complex is OKT3. In some embodiments, the ligandthat binds a protein associated with the TCR complex comprises an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 22. In some embodiments, the ligandthat binds the protein associated with the TCR complex is F6A. In someembodiments, the ligand that binds the protein associated with the TCRcomplex comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity with SEQ ID NO: 24. In someembodiments, the ligand that binds the protein associated with the TCRcomplex is L2K. In some embodiments, the ligand that binds the proteinassociated with the TCR complex comprises an amino acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withSEQ ID NO: 26. In some embodiments, the protein associated with the TCRcomplex is CD3. In some embodiments, the target-specific ligandselectively binds a tumor antigen. In some embodiments, thetarget-specific ligand is a designed ankyrin repeat (DARPin)polypeptide, or a single chain variable fragment (scFv). In someembodiments, the target-specific ligand selectively binds a CD19antigen, a HER2 antigen, or a BCMA antigen. In some embodiments, thetarget-specific ligand selectively binds a HER-2 antigen comprises anantigen binding domain of an antibody selected from Trastuzumab,Pertuzumab, Lapatinib, Neratinib, Ado-trastuzmab Emtansine, Gancotamab,Margetuximab, Timigutuzumab, and Ertumaxomab. In some embodiments, thetarget-specific ligand selectively binds a BCMA antigen comprises anantigen binding domain of an antibody selected from Belantamabmafodotin, and GSK2857916. In some embodiments, the target-specificligand comprises an amino acid sequence having at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36, SEQ IDNO: 8 or SEQ ID NO: 34. In some embodiments, the T cell receptorsignaling domain polypeptide comprises a cytosolic domain and atransmembrane domain. In some embodiments, the cytosolic domain is a CD4cytosolic domain and the transmembrane domain is a CD4 transmembranedomain, or wherein the cytosolic domain is a CD8 cytosolic domain andthe transmembrane domain is a CD8 transmembrane domain. In someembodiments, the nucleic acid sequences further comprise a leadersequence. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50. Insome embodiments, the CD3 is of a TCR complex on a cell expressing thesecond polynucleotide. In some embodiments, the binding of the CD3induces activation of a cell expressing the second polynucleotide. Insome embodiments, the Tri-TAC comprises a nucleic acid sequence havingat least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withSEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, or SEQ ID NO: 61. In some embodiments, the Tri-TACcomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity with SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 68, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ IDNO: 62. In some embodiments, the nucleic acid sequence does not encode aco-stimulatory domain. In some embodiments, the nucleic acid sequencedoes not encode an activation domain.

Disclosed herein, in certain embodiments, are nucleic acid sequencesencoding a Trifunctional T cell-antigen coupler (Tri-TAC) comprising:(a) a first polynucleotide encoding a target-specific ligand; (b) asecond polynucleotide encoding a ligand that binds a protein associatedwith a TCR complex; and (c) a third polynucleotide encoding a T cellreceptor signaling domain polypeptide; wherein the nucleic acid sequencefurther comprises a leader sequence, and wherein component encoded by(a), component encoded by (b), and component encoded by (c) are fuseddirectly to each other, or joined by at least one linker. In someembodiments, the target-specific ligand selectively binds a tumorantigen. In some embodiments, the target-specific ligand is a designedankyrin repeat (DARPin) polypeptide, or a single chain variable fragment(scFv). In some embodiments, the target-specific ligand selectivelybinds a CD19 antigen, a HER2 antigen, or a BCMA antigen. In someembodiments, the target-specific ligand selectively binds a HER-2antigen comprises an antigen binding domain of an antibody selected fromTrastuzumab, Pertuzumab, Lapatinib, Neratinib, Ado-trastuzmab Emtansine,Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab. In someembodiments, the target-specific ligand selectively binds a BCMA antigencomprises an antigen binding domain of an antibody selected fromBelantamab mafodotin, and GSK2857916. In some embodiments, thetarget-specific ligand comprises an amino acid sequence having at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity with SEQ IDNO: 36, SEQ ID NO: 8, SEQ ID NO: 34, SEQ ID NO: 52, or SEQ ID NO: 54. Insome embodiments, the ligand that binds the protein associated with theTCR complex is selected from UCHT1, UCHT1 (Y182T), huUCHT1, huUCHT1(Y177T), OKT3, F6A, or L2K. In some embodiments, the ligand that binds aprotein associated with the TCR complex has an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identitywith SEQ ID NO: 14, SEQ ID NO: 72, SEQ ID NO: 44, SEQ ID NO: 46, SEQ IDNO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. In some embodiments, theprotein associated with the TCR complex is CD3. In some embodiments, theT cell receptor signaling domain polypeptide comprises a cytosolicdomain and a transmembrane domain. In some embodiments, the cytosolicdomain is a CD4 cytosolic domain and the transmembrane domain is a CD4transmembrane domain, or wherein the cytosolic domain is a CD8 cytosolicdomain and the transmembrane domain is a CD8 transmembrane domain. Insome embodiments, the leader sequence comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identitywith SEQ ID NO: 6, SEQ ID NO: 48, or SEQ ID NO: 50. In some embodiments,the component encoded by (a) and the component encoded by (b) aredirectly fused and joined to the component encoded by (c) by a linker.In some embodiments, the component encoded by (b) and the componentencoded by (c) are directly fused and joined to the component encoded by(a) by a linker. In some embodiments, the at least one linker is a G₄Sflexible linker (SEQ ID NO: 73), a large protein domain, a long helixstructure, or a short helix structure. In some embodiments, the at leastone linker has an amino acid sequence having at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity with SEQ ID NO: 12 (G₄S flexiblelinker (“G₄S” disclosed as SEQ ID NO: 73)), SEQ ID NO: 32 (large proteindomain), SEQ ID NO: 30 (long helix structure), or SEQ ID NO: 28 (shorthelix structure). In some embodiments, the CD3 is of a TCR complex on acell expressing the second polynucleotide. In some embodiments, thebinding of the CD3 induces activation of a cell expressing the secondpolynucleotide. In some embodiments, the Tri-TAC comprises a nucleicacid sequence having at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61. In someembodiments, the Tri-TAC comprises an amino acid sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withSEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, or SEQ ID NO: 62. In some embodiments, the nucleicacid sequence does not encode a co-stimulatory domain. In someembodiments, the nucleic acid sequence does not encode an activationdomain.

Disclosed herein, in certain embodiments, are polypeptides encoded bythe nucleic acid sequence disclosed herein.

Disclosed herein, in certain embodiments, are vector constructscomprising: (a) a nucleic acid sequence disclosed herein; and (b) apromoter functional in a mammalian cell.

Disclosed herein, in certain embodiments, are T cells comprising thenucleic acid sequence disclosed herein.

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising the T cell disclosed herein, and apharmaceutically acceptable excipient.

Disclosed herein, in certain embodiments, are methods of treating acancer in an individual in need thereof, comprising administering to theindividual a pharmaceutical composition disclosed herein. In someembodiments, the subject is a mammal. In some embodiments, the cancer isa solid cancer or a liquid cancer. In some embodiments, the cancer is alung cancer, a breast cancer, multiple myeloma, glioblastoma, gastriccancer, ovarian cancer, stomach cancer, colorectal cancer, urothelialcancer, endometrial cancer, or a colon cancer. In some embodiments, thecancer comprises a CD19 expressing cancer cell. In some embodiments, thecancer is a B cell malignancy. In some embodiments, the cancer is B celllymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), or Non-Hodgkins Lymphoma. In some embodiments, thecancer comprises a HER-2 expressing cancer cell. In some embodiments,the cancer is breast cancer, bladder cancer, pancreatic cancer, ovariancancer, or stomach cancer. In some embodiments, the cancer comprises aBCMA expressing cancer cell. In some embodiments, the cancer isleukemia, lymphoma, or multiple myeloma. In some embodiments, thepharmaceutical composition is administered to the individualtransarterially, subcutaneously, intradermally, intratumorally,intranodally, intrameduliary, intramuscularly, intravenously orintraperitoneally. In some embodiments, the pharmaceutical compositionis in a unit dose form. In some embodiments, the pharmaceuticalcomposition is comprises about 0.5-2×10⁹ T cells. In some embodiments,the pharmaceutical composition is administered daily, weekly, bi-weekly,monthly, bi-month or yearly.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A is a schematic of natural T-cell activation.

FIG. 1B is a schematic of CAR based T-cell activation.

FIG. 1C is a schematic of a trifunctional-T cell-antigen coupler(Tri-TAC) based T cell activation.

FIG. 1D is a schematic of natural T-cell activation.

FIG. 1E is a schematic of CAR based T-cell activation.

FIG. 1F is a schematic of Tri-TAC based T cell activation.

FIG. 2A is a schematic of a Tri-TAC configuration with the UCHT1 domainbeing centered between the trans-membrane domain (TM) and the antigenbinding domain.

FIG. 2B is a schematic of a Tri-TAC configuration in which the UCHT1domain is N-terminal, followed by the antigen binding domain and thetrans-membrane domain.

FIG. 2C is a schematic of a Tri-TAC molecule with a generic antigenbinding domain and a UCHT1 domain.

FIG. 3A is a schematic of a Tri-TAC molecule with a generic antigenbinding domain.

FIG. 3B is a schematic of a Tri-TAC with an anti-HER-2 DARPin antigenbinding domain.

FIG. 3C is a schematic of a Tri-TAC with an anti-CD19 scFv antigenbinding domain.

FIG. 3D is a schematic of a Tri-TAC with an anti-BCMA scFv antigenbinding domain.

FIG. 3E is a schematic of a Tri-TAC molecule with the Anti-HER-2 DARPinantigen binding domain.

FIG. 3F is a schematic of a Tri-TAC molecule with the Anti-BCMA scFvantigen binding domain.

FIG. 4A-FIG. 4D exemplify T cells engineered with a Tri-TAC or aCD28-based CAR directed against HER-2 using a DARPin. FIG. 4Aexemplifies the surface expression of the Tri-TAC and CAR compared to Tcells that express no chimeric receptor. FIG. 4B exemplifies growth ofthree cell populations. FIG. 4C-FIG. 4D exemplify the percentage ofengineered cells positive for various T cell activation markersfollowing stimulation with antigen.

FIG. 5 illustrates a model of the CD19-TAC protein structure.

FIG. 6A-FIG. 6J illustrate receptor surface expression and activation ofvarious anti-HER-2 DARPin Tri-TAC controls. T cells were engineered witha Tri-TAC variant that lacks the targeting element (-DARPin), a Tri-TACvariant that lacks UCHT1 (-UCHT1), or the full-length Tri-TAC. FIG. 6A,FIG. 6D, FIG. 6G illustrate T cell transduction and Her2 binding ability(left); FIG. 6B, FIG. 6E, FIG. 6H degranulation (middle) and FIG. 6C,FIG. 6F, FIG. 6I cytokine production (right). FIG. 6J illustrates thatonly full length anti-HER-2 DARPin Tri-TAC is able to elicit a cytotoxicresponse.

FIG. 7A-FIG. 7C illustrate anti-tumor activity, toxicity, and cytokineproduction of T cells engineered with either the anti-HER-2 DARPinTri-TAC or the anti-HER-2 DARPin CD28-based CAR. Mice bearingestablished OVCAR-3 tumors were treated with T cells engineered with theanti-HER-2 DARPin Tri-TAC or the anti-HER-2 DARPin CAR. FIG. 7Aexemplifies the change in tumor growth relative to the day of T cellinfusion (day 35). FIG. 7B exemplifies the change in weight, a measureof toxicity, in the same mice. FIG. 7C illustrates cytokineconcentrations in serum of mice on day 7 post T-cell infusion.

FIG. 8A-FIG. 8H illustrate Tri-TACs designed with various alternativesto the UCHT1 scFv-CD3 recruitment domain. FIG. 8A provides a schematicrepresentation of TAC receptor constructs utilizing the anti-HER-2DARPin, paired with either the UCHT1 or OKT3 anti-CD3 scFv. FIG. 8Billustrates HER-2 TAC surface expression of CD8+ NGFR+ (left) or CD4+NGFR+ T cells (right). FIG. 8C, FIG. 8C1 illustrate cytokine productionby HER-2-specific TAC-T cells stimulated with antigen-positive SK-OV-3tumor cells. FIG. 8D illustrates killing of SK-OV-3 tumor cells by HER-2TAC and vector control (vector only carrying tNGFR) T cells. Vectorcontrol T cells (circles) are compared against HER-2-specific TAC-Tcells bearing UCHT1 (square) or OKT3 (triangle). FIG. 8E provides aschematic representation of TAC receptor constructs utilizing theanti-CD19 scFv, paired with either huUCHT1, F6A, or L2K anti-CD3 scFv.FIG. 8F illustrates CD19-TAC surface expression of CD8+ NGFR+ (left) orCD4+ NGFR+ T cells (right). FIG. 8G, FIG. 8G1 illustrate cytokineproduction by CD19-specific TAC-T cells stimulated with antigen-positiveRaji tumor cells. Cytokine producing cells are compared from TAC-T cellsbearing huUCHT1 (square), F6A (triangle), or L2K (diamond). FIG. 8Hillustrates killing of NALM-6 tumor cells by CD19 TAC and vector control(vector only carrying tNGFR) T cells. Vector control T cells (circles)are compared against CD19-specific TAC-T cells bearing huUCHT1 (square),F6A (triangle), or L2K (diamond).

FIG. 9A-FIG. 9H illustrates the effect of various anti-CD3 scFv on TCRsurface expression. FIG. 9A, FIG. 9E illustrate TCR surface expressionof T cells engineered with either control vector (tNGFR), UCHT1, or OKT3TAC variants. FIG. 9B, FIG. 9F illustrate that T cells engineered withOKT3-TAC have significantly reduced TCR surface expression relative toUCHT1-TAC. FIG. 9C, FIG. 9G illustrate TCR surface expression of T cellsengineered with control vector (tNGFR), huUCHT1, F6A or L2K TACvariants. FIG. 9D, FIG. 9H illustrates that T cells engineered with L2KTAC have significantly reduced TCR surface expression relative tohuUCHT1-TAC.

FIG. 10A-FIG. 10B illustrate connector domain variants. The domain theconnecting antigen binding domain with the TCR recruitment domain istermed the connector domain. FIG. 10A provides schematics of TACvariants with different connector domains: (i) a flexible connector,(ii) a large domain connector (constructed from domains 3 and 4 derivedfrom the extracellular CD4 domain), (iii) a long helical connector, and(iv) a short helical connector. FIG. 10B provides exemplary amino acidsequence of the domains represented in FIG. 10A. (SEQ ID NOS 69, 28, 30,and 32, respectively, in order of appearance)

FIG. 11A-FIG. 11E illustrate exemplary in vitro parameters of CD19 TACengineered with different connector variants. FIG. 11A illustrates TACvariant surface expression in both CD4 and CD8 cells. FIG. 11Billustrates surface expression of TAC comprising flexible connectorsrelative to TAC comprising helical or large domain connectors. FIG. 11Cillustrates overall transduction of TAC comprising alternativeconnectors relative to the flexible connector. FIG. 11D, FIG. 11Eillustrate relative cell reactivity to antigen positive Raji cells.

FIG. 12A illustrates in vitro cytotoxicity of BCMA Tri-TAC variantsengineered with different connectors. FIG. 12B illustrates in vivo tumorcontrol of BCMA Tri-TAC variants engineered with the flexible connectorcompared to the short helical connector.

FIG. 13A-FIG. 13C illustrate properties of CD8α Tri-TAC scFv anti HER-2,and CD8α Tri-TAC DARPin anti-HER-2. FIG. 13A, FIG. 13C illustratesurface expression. FIG. 13B illustrates cytokine production.

FIG. 14A-FIG. 14D provide schematics of CD8 Tri-TAC variants. The antiHER-2-DARPin is used as an exemplary antigen-binding domain and theUCHT1 CD3 recruitment domain is used as an exemplary recruitment domain.FIG. 14A illustrates a Tri-TAC comprising a CD4 transmembrane andcytosolic domain (left), and comparable regions of a CD8α/CD8βheterodimer (right). Key regions for co-receptor functionality (argininerich domain and CXCP motif) are highlighted. FIG. 14B is a schematic ofa CD8α Tri-TAC comprising a Cysteine to Serine mutation to ensure amonomeric receptor distribution, and a CD8α cytosolic domain. FIG. 14Cis a schematic of a CD8α+Rβ Tri-TAC comprising a Cysteine to Serinemutation to ensure a monomeric receptor distribution, and a chimericCD8α cytosolic domain where the CD8α arginine rich region is replacedwith the CD8β arginine rich region. FIG. 14D is a schematic of aCD8β+Lck Tri-TAC comprising a Cysteine to Serine mutation to ensure amonomeric receptor distribution, and a chimeric CD8β cytosolic domain,where the CD8α CXCP domain, which contains an Lck binding motif, wasadded to the C-terminus of the CD8β cytosolic domain.

FIG. 15A-FIG. 15E illustrate in vitro characterization of CD8 Tri-TACvariants relative to the prototypic Tri-TAC containing CD4 regions. FIG.15A-FIG. 15B illustrate surface expression of CD8-Tri TAC variantsrelative to the prototypic Tri-TAC. FIG. 15C illustrates in vitrocytotoxicity of CD8-Tri TAC variants co-cultured with LOX IMVI (HER-2negative) or A549, SKOV3, SKBR3 or MBA MB 231 (HER-2 positive). FIG. 15Dillustrates cell division of T cells engineered with either the CD8Tri-TAC variants or the prototypic Tri-TAC. FIG. 15E illustrates TCRsurface expression of engineered T cells comprising CD8 Tri-TAC variantsor the prototypic Tri-TAC.

FIG. 16 illustrates various Tri-TACs.

FIG. 17 illustrates TAC-CD19 insert in a pCCL lentiviral vector. FIG. 17illustrates the various domains of a TAC-CD19 (a CD8a leader, FMC63scFv, Myc Tag, huUCHT1 Y177T mutant and a truncated CD4 anchoringco-receptor domain).

FIG. 18 illustrates the in vivo efficacy of TAC-CD19 generated fromdifferent donors.

FIG. 19A-FIG. 19C illustrates an in vitro example of TAC-CD19cytotoxicity against the tumor lines. FIG. 19A NALM-6 (acutelymphoblastic leukemia), FIG. 19B Jeko-1(Mantle Cell Lymphoma) and FIG.19C Raji (Burkitt's lymphoma).

FIG. 19D illustrates the schematic of 3 different in vivo tumor modelsin NRG mice.

FIG. 19E-FIG. 19G illustrate in vivo efficacy of CD19-TAC in NALM-6(acute lymphoblastic leukemia) FIG. 19E, Jeko-1(Mantle Cell Lymphoma)FIG. 19F, and Raji (Burkitt's lymphoma) FIG. 19G.

FIG. 20A illustrates the experimental set up of TAC-CD19 treated micewith NALM-6 tumor. Following successful treatment mice are thenre-challenged with either NALM-6 (CD19 positive) or KMS11 (CD19negative) tumor cells.

FIG. 20B illustrates in vivo efficacy of mice treated with TAC-CD19.

FIG. 21A illustrates the experimental design of evaluating dose regimeand dosing impact on efficacy and cell expansion.

FIG. 21B illustrates in vivo survival of NALM-6 bearing mice treatedwith either a single or split dose of TAC-CD19.

FIG. 22A-FIG. 22B illustrate an experimental setup and data with regardto in vivo expansion of TAC-CD19 following a split dose administration.FIG. 22A illustrates the gating strategy used to identify T cells inmouse blood. FIG. 22B illustrates in vivo results of T cell expansion inblood.

FIG. 23A-FIG. 23C illustrate long term in vivo studies of TAC-CD19 inmice. FIG. 23A illustrates an experimental protocol of NALM-6 bearingmice being treated with various controls and TAC-CD19 at two doselevels. FIG. 23B illustrates in vivo efficacy of control vs two doselevels of TAC-CD19 treatment groups. FIG. 23C illustrates long termsurvival of low dose TAC-CD19 treated mice.

FIG. 24 illustrates clinical chemistry analysis results from micetreated with TAC-CD19 or non-transduced T cells.

FIG. 25 illustrates human cytokine released in mice blood followingtreatment with TAC-CD19 or non-transduced T cells.

FIG. 26A-FIG. 26C illustrates efficacy of BCMA-TAC in differentconfigurations. FIG. 26A illustrates an experimental design. FIG. 26Billustrates various controls and test articles. FIG. 26C illustrates invivo efficacy of various TAC constructs. FIG. 26A-FIG. 26C disclose“G₄S” as SEQ ID NO: 73.

FIG. 27 illustrates that TACs proliferate when encountering antigen oncells, but not when the antigen is presented on artificial beads; butCARs proliferate irrespective if antigens are presented on beads orcells.

FIG. 28A-FIG. 28B illustrate TAC engineered T cells expand in vivo andprovide long term protection, indicating cell persistence in a model ofmyeloma. FIG. 28A-FIG. 28B illustrate BCMA-TAC T cells reject multiplemyeloma tumors in a KMS-11 xenograft model engineered with NanoLuc (KMS11-NanoLuc) (BCMA^(pos)). Following tumor engraftment mice were treatedwith BCMA TAC-T cells (carrying Firefly Luciferase). TAC-T cells expandsignificantly following administration. This correlates with tumorregression. Treated mice were resistant to tumor rechallenge indicatinglong term persistence of TAC-T cells.

FIG. 29 illustrates human cytokine released in mice blood followingtreatment with TAC-CD19 or non-transduced T cells.

DETAILED DESCRIPTION

Cancer is a major health challenge, with over 150,000 cases of cancerexpected to be diagnosed in Canada alone. While patients with earlystage disease are sometimes treated effectively by conventionaltherapies (surgery, radiation, chemotherapy), few options are availableto patients with advanced disease, and those options are typicallypalliative in nature.

Active immunotherapy seeks to employ the patient's immune system toclear tumors and offers an option to patients who have failedconventional therapies. Generally, this treatment involves infusingpatients with large numbers of tumor-specific T cells. This approach hasproven to be successful in early phase clinical trials for a number ofdiseases, including melanoma, myeloma, leukemia, lymphoma and synovialsarcoma. As a specific example, several clinical studies havedemonstrated that immunotherapy with T cells are curative in patientswith advanced melanoma, confirming the utility of this approach.Additionally, patients suffering from chronic lymphocytic leukemia (CLL)and acute lymphoblastic leukemia (ALL) have also been effectivelytreated and cured with T cell immunotherapy.

A key challenge facing the clinical application of adoptive T celltherapy is the source of the T cells. Typically, T cells isolated from atumor-bearing patient are grown to large numbers ex vivo and areadministered back into the patient to induce a robust anti-tumor immuneresponse. Tumor specificity is achieved by either: (i) isolatingnaturally-occurring tumor-specific T cells from the patient; or (ii)engineering bulk T cells from the peripheral blood to expresstumor-specific receptors. Naturally occurring tumor-specific T cells arerare and isolating such cells in therapeutic quantities from cancerpatients is a laborious and costly procedure. In contrast, it isbecoming more efficient to engineer readily available peripheral T cellswith tumor-specific receptors through genetic manipulation. Techniqueshave been developed for this engineering process, which are clinicallyviable, and several clinical trials have demonstrated the feasibilityand efficacy of genetically-engineered T cells for the treatment ofcancer.

To this point, most engineered T cell therapies involving geneticmodification of the T cells yield: (i) forced expression of T cellreceptor (TCR); or (ii) a chimeric antigen receptor (CAR) specific forantigen targets on the tumor. To date, the chimeric antigen receptorsused for engineering T cells consist of: (i) a targeting domain, usuallya single-chain fragment variable (scFv); (ii) a transmembrane domain;and (iii) a cytosolic domain that contains signaling elements from the Tcell receptor and associated proteins. Such chimeric antigen receptorshave also been referred to as “T-body” or “Chimeric Immune Receptor”(CIR), but currently, most researchers use the term “CAR”. One advantageof the CAR approach is that it allows any patient's immune cells to betargeted against any desirable target in a major histocompatibilitycomplex (MHC) independent manner. This is appealing as MHC presentationis often defective in tumor cells.

CARs are considered in modular terms and scientists have spentconsiderable time investigating the influence of different cytoplasmicsignaling domains on CAR function. Conventional CARs generally share twomain components: (i) the CD3 zeta cytoplasmic domain, which containsimmunotyrosine activation motifs (ITAMs) critical for T cell activation;and (ii) components of costimulatory receptors that trigger importantsurvival pathways such as the Akt pathway.

The first-generation CARs employed a single signaling domain from eitherCD3ζ or FcεRIγ. Second-generation CARs combined the signaling domain ofCD3ζ with the cytoplasmic domain of costimulatory receptors from eitherthe CD28 or TNFR family of receptors. Most CAR-engineered T cells thatare currently being tested in the clinic employ second-generation CARswhere CD3ζ is coupled to the cytoplasmic domain of either CD28 or CD137.These second generation CARs have demonstrated anti-tumor activity inCD19-positive tumors. Third-generation CARs combined multiplecostimulatory domains, but there is concern that third-generation CARsmay lose antigen-specificity.

While CAR-engineered T cells have shown considerable promise in clinicalapplication, they rely on a synthetic method for replacing the nativeactivation signal that is provided by the T cell receptor (TCR). Sincethis synthetic receptor does not deliver all of the signaling componentsassociated with the TCR (ex. ITAMs on CD3γ, CD3δ, CD3ε), it remainsunclear whether the T cells are optimally activated by the CAR or howthe CAR activation affects T cell differentiation (ex. progression tomemory). Furthermore, since the CAR signaling domains are disconnectedfrom their natural regulatory partners by the very nature of the CARstructure, there is an inherent risk that CARs may lead to a low-levelof constitutive activation, which could result in off-target toxicities.Therefore, the synthetic nature of the prototypic CAR may disruptcanonical mechanisms that limit TCR activation, and may underpin thesevere toxicity often associated with therapeutic doses of conventionalCAR T cells.

Given these limitations, it is preferable to re-direct T cells to attacktumors via their natural TCR. To this end, a class of recombinantproteins termed “Bispecific T-cell Engagers” (BiTEs) has been created.These proteins employ bispecific antibody fragments to crosslink T-cellTCR receptors with target antigens. This leads to efficient T-cellactivation, triggering cytotoxicity. Similarly, bispecific antibodieshave been generated that accomplish this goal and some scientists havesimply linked anti-CD3 antibodies to tumor-specific antibodies employingchemical linkage. While these bispecific proteins have demonstrated someactivity in vitro, GMP production, short biological half-lives, andlimited bioavailability represent significant challenges to thesuccessful use of these molecules in cancer treatment. Additionally,these molecules also fail to properly recapitulate natural TCR signalingbecause they do not engage the TCR co-receptors (CD8 and CD4).

In view of the above, a need remains for chimeric receptors withenhanced activity and safety.

An alternate chimeric receptor, termed a Trifunctional T cell AntigenCoupler (Tri-TAC or TAC) receptor, has been developed which employs adistinct biology to direct the T cell to attack tumors. While the CAR isa fully synthetic receptor that stitches together components of T cellreceptor (TCR) signaling complex, the TAC receptor re-directs the TCRtowards tumor targets and recapitulates the native TCR signalingstructure. For example, in some embodiments, the TACs disclosed hereinactivate natural Major Histocompatibility complex (MHC) signalingthrough the T-cell receptor (TCR), while retaining MHC-unrestrictedtargeting. Further, the TACs disclosed herein recruit the T-CellReceptor (TCR) in combination with co-receptor stimulation. Moreover, insome embodiments, Tri-TACs disclosed herein show enhanced activity andsafety.

Certain Terminology

The term “T cell” as used herein refers to a type of lymphocyte thatplays a central role in cell-mediated immunity. T cells, also referredto as T lymphocytes, are distinguished from other lymphocytes, such as Bcells and natural killer cells, by the presence of a T-cell receptor(TCR) on the cell surface. There are several subsets of T cells withdistinct functions, including but not limited to, T helper cells,cytotoxic T cells, memory T cells, regulatory T cells and natural killerT cells.

The term “T cell antigen coupler” or TAC is used interchangeably with“trifunctional T cell antigen coupler” or Tri-TAC and refers to anengineered nucleic acid construct or polypeptide, that when expressed ona T cell, helps to facilitate the targeting of the T cell to aparticular antigen. In some embodiments, the TAC comprises (a) atarget-specific ligand, (b) a ligand that binds a protein associatedwith a T cell receptor (TCR) complex, and (c) a T cell receptorsignaling domain.

The term “polynucleotide” and/or “nucleic acid sequence” and/or “nucleicacid” as used herein refers to a sequence of nucleoside or nucleotidemonomers consisting of bases, sugars and intersugar (backbone) linkages.The term also includes modified or substituted sequences comprisingnon-naturally occurring monomers or portions thereof. The nucleic acidsequences of the present application may be deoxyribonucleic acidsequences (DNA) or ribonucleic acid sequences (RNA) and may includenaturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The sequences may also contain modified bases.Examples of such modified bases include aza and deaza adenine, guanine,cytosine, thymidine and uracil; and xanthine and hypoxanthine. Thenucleic acids of the present disclosure may be isolated from biologicalorganisms, formed by laboratory methods of genetic recombination orobtained by chemical synthesis or other known protocols for creatingnucleic acids.

The term “isolated polynucleotide” or “isolated nucleic acid sequence”as used herein refers to a nucleic acid substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors, or other chemicals when chemically synthesized.An isolated nucleic acid is also substantially free of sequences whichnaturally flank the nucleic acid (i.e. sequences located at the 5′ and3′ ends of the nucleic acid) from which the nucleic acid is derived. Theterm “nucleic acid” is intended to include DNA and RNA and is eitherdouble stranded or single stranded, and represents the sense orantisense strand. Further, the term “nucleic acid” includes thecomplementary nucleic acid sequences.

The term “recombinant nucleic acid” or “engineered nucleic acid” as usedherein refers to a nucleic acid or polynucleotide that is not found in abiological organism. For example, recombinant nucleic acids may beformed by laboratory methods of genetic recombination (such as molecularcloning) to create sequences that would not otherwise be found innature. Recombinant nucleic acids may also be created by chemicalsynthesis or other known protocols for creating nucleic acids.

The term “polypeptide” or “protein” as used herein describes a chain ofamino acids. A polypeptide or protein of this disclosure is a peptide,which usually describes a chain of amino acids. The term protein as usedherein also describes a large molecule comprising one or more chains ofamino acids and, in some embodiments, is a fragment or domain of aprotein or a full length protein. Furthermore, as used herein, the termprotein either refers to a linear chain of amino acids or to a chain ofamino acids that has been processed and folded into a functionalprotein. The protein structure is divided into four distinct levels: (1)primary structure—referring to the sequence of amino acids in thepolypeptide chain, (2) secondary structure—referring to the regularlocal sub-structures on the polypeptide backbone chain, such as α-helixand β-sheets, (3) tertiary structure—referring to the three-dimensionalstructure if monomeric and multimeric protein molecules, and (4)quaternary structure—referring to the three-dimensional structurecomprising the aggregation of two or more individual polypeptide chainsthat operate as a single functional unit. The proteins of the presentdisclosure, in some embodiments, are obtained by isolation andpurification of the proteins from cells where they are producednaturally, by enzymatic (e.g., proteolytic) cleavage, and/orrecombinantly by expression of nucleic acid encoding the proteins orfragments of this disclosure. The proteins and/or fragments of thisdisclosure, in some embodiments, is obtained by chemical synthesis orother known protocols for producing proteins and fragments.

The term “isolated polypeptide” refers to a polypeptide substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, single chain antibodies, chimericantibodies, and antibody fusions. The antibody may be from recombinantsources and/or produced in transgenic animals. The term “antibodyfragment” as used herein is intended to include without limitations Fab,Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof, multispecific antibody fragments and DomainAntibodies.

The term “vector” as used herein refers to a polynucleotide that is usedto deliver a nucleic acid to the inside of a cell. In some embodiments,a vector is an expression vector comprising expression control sequences(for example, a promoter) operatively linked to a nucleic acid to beexpressed in a cell. Vectors known in the art include, but are notlimited to, plasmids, phages, cosmids and viruses.

The term “tumor antigen” or “tumor associated antigen” as used hereinrefers to an antigenic substance produced in tumor cells that triggersan immune response in a host (e.g. which is presented by MHC complexes).In some embodiments, a tumor antigen is on the surface of a tumor cell.

The term “T cell receptor” or TCR as used herein refers to a complex ofintegral membrane proteins that participates in the activation of Tcells in response to the binding of an antigen. The TCR is adisulfide-linked membrane-anchored heterodimer normally consisting ofthe highly variable alpha (α) and beta (β) chains expressed as part of acomplex with the invariant CD3 (cluster of differentiation 3) chainmolecules. T cells expressing this receptor are referred to as α:β (orαβ) T cells, though a minority of T cells express an alternate receptor,formed by variable gamma (γ) and delta (δ) chains, referred as γδ Tcells. CD3 is a protein complex composed of four distinct chains. Inmammals, the complex contains a CD3γ chain, a CD3δ chain, two CD3εchains and two CD3ζ chains.

As used herein, the term “transmembrane and cytosolic domain” refers toa polypeptide that comprises a transmembrane domain and a cytosolicdomain of a protein associated with the T cell receptor (TCR) complex.In some embodiments, such transmembrane and cytosolic domain mayinclude, but is not limited to, protein domains that (a) associate withthe lipid raft and/or (b) bind Lck.

A “TCR co-receptor” as used herein, refers to a molecule that assiststhe T cell receptor (TCR) in communicating with an antigen-presentingcell and may be considered part of the first signal that leads to theactivation of the TCR. Examples of TCR co-receptors include, but are notlimited to, CD4, LAG3, and CD8.

A “TCR co-stimulator” as used herein, refers to a molecule that enhancesthe response of a T cell to an antigen and may be considered as thesecond signal that leads to the activation of the TCR. Examples of TCRco-stimulators include, but are not limited to, ICOS, CD27, CD28, 4-1BB(CD 137), OX40 (CD134), CD30, CD40, lymphocyte fiction-associatedantigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand thatspecifically binds CD83.

A “TCR co-inhibitor” or “checkpoint receptor” as used herein, refers toa molecule that inhibits the response of a T cell to an antigen.Examples of TCR co-inhibitors include, but are not limited to, PD-1,TIM3, LAG-3, TIGIT, BTLA, CD160, and CD37.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and in some embodiments, refer to anymammalian subject for whom diagnosis, treatment, or therapy is desired,particularly humans. “Mammal” for purposes of treatment refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and laboratory, zoo, sports, or pet animals, such as dogs,horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guineapigs, monkeys etc. In some embodiments, the mammal is human. None ofthese terms require the supervision of medical personnel.

As used herein, the terms “treatment,” “treating,” and the like, in someembodiments, refer to administering an agent, or carrying out aprocedure, for the purposes of obtaining an effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of affecting apartial or complete cure for a disease and/or symptoms of the disease.“Treatment,” as used herein, may include treatment of a disease ordisorder (e.g. cancer) in a mammal, particularly in a human, andincludes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease. Treating may refer to anyindicia of success in the treatment or amelioration or prevention of acancer, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms; or making the diseasecondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. The treatment or amelioration of symptoms is based on oneor more objective or subjective parameters; including the results of anexamination by a physician. Accordingly, the term “treating” includesthe administration of the compounds or agents of the present inventionto prevent, delay, alleviate, arrest or inhibit development of thesymptoms or conditions associated with diseases (e.g. cancer). The term“therapeutic effect” refers to the reduction, elimination, or preventionof the disease, symptoms of the disease, or side effects of the diseasein the subject.

As used herein, singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “an antibody” includes a plurality of antibodiesand reference to “an antibody” in some embodiments includes multipleantibodies, and so forth.

As used herein, all numerical values or numerical ranges include wholeintegers within or encompassing such ranges and fractions of the valuesor the integers within or encompassing ranges unless the context clearlyindicates otherwise. Thus, for example, reference to a range of 90-100%,includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%,91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%,etc., and so forth. In another example, reference to a range of 1-5,000fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc.,2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

“About” a number, as used herein, refers to range including the numberand ranging from 10% below that number to 10% above that number. “About”a range refers to 10% below the lower limit of the range, spanning to10% above the upper limit of the range.

“Percent (%) identity” refers to the extent to which two sequences(nucleotide or amino acid) have the same residue at the same positionsin an alignment. For example, “an amino acid sequence is X % identicalto SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQID NO:Y and is elaborated as X % of residues in the amino acid sequenceare identical to the residues of sequence disclosed in SEQ ID NO: Y.Generally, computer programs are employed for such calculations.Exemplary programs that compare and align pairs of sequences, includeALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988;Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN,or GCG (Devereux et al., 1984).

As used herein, the term “selective binding” refers to the higheraffinity with which a molecule (e.g. protein such as a target-bindingligand of TAC) binds its target molecule (e.g. target antigen such asHER-2, BCMA, or CD19) over other molecules.

T Cell Antigen Coupler (Tri-TAC or TAC)

Disclosed herein, in certain embodiments, are nucleic acids encoding aTrifunctional T cell-antigen coupler (Tri-TAC). In some embodiments, thenucleic acids encoding a Tri-TAC comprises: (a) a first polynucleotideencoding a target-specific ligand; (b) a second polynucleotide encodinga ligand that binds a TCR complex; and (c) a third polynucleotideencoding a transmembrane domain and cytosolic domain. In someembodiments, the nucleic acids encoding a Tri-TAC do not encode aco-stimulatory domain. In some embodiments, the nucleic acids encoding aTri-TAC do not encode a co-activation domain.

Target-Specific Ligand

The target-specific ligand, also referred to as an antigen bindingdomain, refers to any substance or molecule that binds, directly orindirectly, to a target cell. In some embodiments, the target specificligand binds to an antigen on the target cell. In some embodiments, atarget cell is a cell associated with a disease state, including, butnot limited to, cancer, hematologic malignancy, large B-cell lymphoma,diffuse large B-cell lymphoma, primary mediastinal B cell lymphoma, highgrade B-cell lymphoma, or large B cell lymphoma arising from follicularlymphoma. In some embodiments, a target cell is a tumor cell. In someembodiments, a target-specific ligand binds to a tumor antigen or tumorassociated antigen on a tumor cell. In some embodiments, the targetantigen is a tumor antigen. In some embodiments, the tumor antigen whenproteinaceous is a sequence of 8 or more amino acids up to the fullprotein. In some embodiments, the tumor antigen is any number of aminoacids in between 8 and the full length protein which comprises at leastone antigenic fragment of the full length protein that is presented in aMajor Histocompatibility Complex (MHC). Examples of tumor antigensinclude, but are not limited to, CD19, HER-2 (erbB-2), B-cell maturationantigen (BCMA), alphafetoprotein (AFP), carcinoembryonic antigen (CEA),CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase,melanoma-associated antigen (MAGE), prostate-specific antigen (PSA),glioma-associated antigen, β-human chorionic gonadotropin,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, survivin andtelomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M,neutrophil elastase, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-Ireceptor and mesothelin.

In some embodiments, the target-specific ligands include, but are notlimited to, antibodies and fragments thereof, for example single chainantibodies such as single-chain antibodies (scFvs), single domainantibodies, peptides, peptidomimetics, proteins, glycoproteins, orproteoglycans that bind to the target cell and/or antigen. In someembodiments, the target-specific ligands include, but are not limitedto, designed ankyrin repeat proteins (DARPins), lectins, knottins,centryrins, anticalins, or naturally occurring ligands for the tumorantigen, such as growth factors, enzyme substrates, receptors or bindingproteins. In some embodiments, target specific ligands includenon-protein compounds that bind to target cells and/or antigens,including but not limited to carbohydrates, lipids, nucleic acids, orsmall molecules. In some embodiments, a target-specific ligand is adesigned ankyrin repeat (DARPin) targeted to a specific cell and/orantigen. In some embodiments, a target-specific ligand is a single-chainvariable fragment (ScFv) targeted to a specific cell and/or antigen.

In some embodiments, the tumor antigen is a HER-2 antigen. In someembodiments, the HER-2 specific ligand comprises an antigen bindingdomain of an antibody selected from Trastuzumab, Pertuzumab, Lapatinib,Neratinib, Ado-trastuzmab Emtansine, Gancotamab, Margetuximab,Timigutuzumab, and Ertumaxomab. In some embodiments, the target-specificligand is a DARPin that selectively binds a HER-2 (erbB-2) antigen. Insome embodiments, the target-specific ligand is a DARPin thatspecifically binds a HER-2 (erbB-2) antigen. In some embodiments, theDARPin targeted to HER-2 (erb-2) comprises SEQ ID NO: 7 or SEQ ID NO: 8.

In some embodiments, the first polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 7. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 7. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 7. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 7. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 7. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 7. Insome embodiments, the first polynucleotide comprises a nucleotidesequence of SEQ ID NO: 7.

In some embodiments, the target-specific ligand comprises an amino acidsequence having at least 70% sequence identity with SEQ ID NO: 8. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 75% sequence identity with SEQ ID NO: 8. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 8. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 85% sequence identity with SEQ ID NO: 8. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 90% sequence identity with SEQ ID NO: 8. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 95% sequence identity with SEQ ID NO: 8. Insome embodiments, the target-specific ligand comprises an amino acidsequence of SEQ ID NO: 8.

In some embodiments, the tumor antigen is a BCMA antigen. In someembodiments, the BCMA specific ligand comprises an antigen bindingdomain of an antibody selected from Belantamab mafodotin, andGSK2857916. In some embodiments, the target-specific ligand is a scFvthat selectively binds BCMA. In some embodiments, the target-specificligand is a scFv that specifically binds BCMA. In some embodiments, thescFv that binds BCMA comprises SEQ ID NO: 33 or SEQ ID NO: 34.

In some embodiments, the first polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 33. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 33. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 33. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 33. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 33. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 33. Insome embodiments, the first polynucleotide comprises a nucleotidesequence of SEQ ID NO: 33.

In some embodiments, the target-specific ligand comprises an amino acidsequence having at least 70% sequence identity with SEQ ID NO: 34. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 75% sequence identity with SEQ ID NO: 34. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 34. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 85% sequence identity with SEQ ID NO: 34. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 90% sequence identity with SEQ ID NO: 34. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 95% sequence identity with SEQ ID NO: 34. Insome embodiments, the target-specific ligand comprises an amino acidsequence of SEQ ID NO: 34.

In some embodiments, the tumor antigen is a CD19 antigen. In someembodiments, the target-specific ligand is a scFv that selectively bindsCD19. In some embodiments, the target-specific ligand is a scFv thatspecifically binds CD19. In some embodiments, the scFv that binds CD19comprises SEQ ID NO: 35 or SEQ ID NO: 36.

In some embodiments, the first polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 35. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 35. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 35. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 35. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 35. Insome embodiments, the first polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 35. Insome embodiments, the first polynucleotide comprises a nucleotidesequence of SEQ ID NO: 35.

In some embodiments, the target-specific ligand comprises an amino acidsequence having at least 70% sequence identity with SEQ ID NO: 36. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 75% sequence identity with SEQ ID NO: 36. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 36. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 85% sequence identity with SEQ ID NO: 36. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 90% sequence identity with SEQ ID NO: 36. Insome embodiments, the target-specific ligand comprises an amino acidsequence having at least 95% sequence identity with SEQ ID NO: 36. Insome embodiments, the target-specific ligand comprises an amino acidsequence of SEQ ID NO: 36.

Ligand that Binds a TCR Complex

In some embodiments, the TAC comprises a ligand that binds a proteinassociated with the TCR complex. In some embodiments, the ligand thatbinds a protein associated with a TCR complex comprises a substance thatbinds, directly or indirectly, to a protein of the TCR. In someembodiments, the ligand that binds a protein associated with a TCRcomplex comprises a substance that selectively binds to a protein of theTCR. In some embodiments, the ligand that binds a protein associatedwith a TCR complex comprises a substance that specifically binds to aprotein of the TCR. Proteins associated with the TCR include, but arenot limited, to the TCR alpha (α) chain, TCR beta (β) chain, TCR gamma(γ) chain, TCR delta (δ) chain, CD3γ chain, CD3δ chain and CD3ε chains.In some embodiments, a ligand that binds a protein associated with theTCR complex is an antibody to the TCR alpha (α) chain, TCR beta (β)chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3γ chain, CD3δ chainand/or CD3ε chain. In some embodiments, the protein associated with aTCR complex is CD3. In some embodiments, the protein associated with aTCR complex is CD3ε. Examples of CD3 antibodies, include, but are notlimited to, for. In some embodiments, the antibody that binds CD3 is asingle chain antibody, for example a single-chain variable fragment(scFv). In some embodiments, the ligand that binds a TCR is anti-CD3antibody, or a fragment thereof, such as muromonab, otelixizumab,teplizumab, visilizumab, CD3-12, MEM-57, 4D10A6, CD3D, or TR66.

In some embodiments, the CD3 is of a TCR complex on a cell expressingthe second polynucleotide. In some embodiments, the binding of the CD3induces activation of a cell expressing the second polynucleotide.

In some embodiments, the ligand that binds a TCR complex is UCHT1, or avariant thereof. In some embodiments, the ligand that binds a TCRcomplex is UCHT1 (SEQ ID NO: 13, SEQ ID NO: 14 or homologs thereof). Insome embodiments, the UCHT1 ligand binds CD3. In some embodiments, theUCHT1 ligand selectively binds CD3. In some embodiments, the UCHT1ligand specifically binds CD3. In some embodiments, the UCHT1 ligandbinds CD3c. In some embodiments, the UCHT1 ligand selectively bindsCD3c. In some embodiments, the UCHT1 ligand specifically binds CD3c. Insome embodiments, the UCHT1 ligand is encoded by SEQ ID NO 13. In someembodiments, the UCHT1 ligand comprises SEQ ID NO 14. In someembodiments, the UCHT1 ligand is mutated. In some embodiments, the UCHT1ligand comprises a Y182T mutation (also referred to as UCHT1 (Y182T))(SEQ ID NO: 71 and SEQ ID NO: 72). In some embodiments, the UCHT1(Y182T) ligand binds CD3. In some embodiments, the UCHT1 (Y182T) ligandselectively binds CD3. In some embodiments, the UCHT1 (Y182T) ligandspecifically binds CD3. In some embodiments, the UCHT1 (Y182T) ligandbinds CD3ε. In some embodiments, the UCHT1 (Y182T) ligand selectivelybinds CD3c. In some embodiments, the UCHT1 (Y182T) ligand specificallybinds CD3ε. In some embodiments, the UCHT1 (Y182T) ligand is encoded bySEQ ID NO 71. In some embodiments, the UCHT1 (Y182T) ligand comprisesSEQ ID NO 72. In some embodiments, the ligand that binds a TCR complexis a humanized UCHT1 (huUCHT1). In some embodiments, the ligand thatbinds a TCR complex is huUCHT1 (SEQ ID NO 43, SEQ ID NO: 44 or homologsthereof). In some embodiments, the huUCHT1 ligand binds CD3. In someembodiments, the huUCHT1 ligand selectively binds CD3. In someembodiments, the huUCHT1 ligand specifically binds CD3. In someembodiments, the huUCHT1 ligand binds CD3ε. In some embodiments, thehuUCHT1 ligand selectively binds CD3ε. In some embodiments, the huUCHT1ligand specifically binds CD3ε. In some embodiments, the huUCHT1 ligandis encoded by SEQ ID NO 43. In some embodiments, the huUCHT1 ligandcomprises SEQ ID NO 44. In some embodiments, the huUCHT1 has a Y177Tmutation (also referred to as huUCHT1 (Y177T)) (SEQ ID NO: 45 and SEQ IDNO: 46). In some embodiments, the huUCHT1 (Y177T) ligand binds CD3. Insome embodiments, the huUCHT1 (Y177T) ligand selectively binds CD3. Insome embodiments, the huUCHT1 (Y177T) ligand specifically binds CD3. Insome embodiments, the huUCHT1 (Y177T) ligand binds CD3ε. In someembodiments, the huUCHT1 (Y177T) ligand selectively binds CD3ε. In someembodiments, the huUCHT1 (Y177T) ligand specifically binds CD3ε. In someembodiments, the huUCHT1 (Y177T) ligand is encoded by SEQ ID NO 45. Insome embodiments, the huUCHT1 ligand comprises SEQ ID NO 46.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 13. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 13. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 13. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 13. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 13. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 13. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 13.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 14. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 14. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 14. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 14. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 14. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 14. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 14.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 71. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 71. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 71. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 71. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 71. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 71. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 71.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 72. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 72. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 72. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 72. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 72. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 72. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 72.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 43. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 43. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 43. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 43. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 43. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 43. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 43.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 44. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 44. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 44. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 44. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 44. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 44. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 44.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 45. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 45. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 45. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 45. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 45. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 45. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 45.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 46. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 46. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 46. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 46. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 46. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 46. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 46.

In some embodiments, the ligand that binds to a CD3 is OKT3. In someembodiments, the OKT3 ligand binds CD3. In some embodiments, the OKT3ligand selectively binds CD3. In some embodiments, the OKT3 ligandspecifically binds CD3. In some embodiments, the OKT3 ligand binds CD3ε.In some embodiments, the OKT3 ligand selectively binds CD3ε. In someembodiments, the OKT3 ligand specifically binds CD3c. In someembodiments, the murine OKT3 ligand is encoded by SEQ ID NO 21. In someembodiments, the OKT3 ligand comprises SEQ ID NO 22.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 21. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 21. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 21. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 21. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 21. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 21. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 21.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 22. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 22. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 22. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 22. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 22. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 22. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 22.

In some embodiments, the ligand that binds to a CD3 is F6A. In someembodiments, the F6A ligand binds CD3. In some embodiments, the F6Aligand selectively binds CD3. In some embodiments, the F6A ligandspecifically binds CD3. In some embodiments, the F6A ligand binds CD3c.In some embodiments, the F6A ligand selectively binds CD3c. In someembodiments, the F6A ligand specifically binds CD3c. In someembodiments, the murine F6A ligand is encoded by SEQ ID NO 23. In someembodiments, the F6A ligand comprises SEQ ID NO 24.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 23. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 23. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 23. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 23. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 23. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 23. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 23.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 24. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 24. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 24. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 24. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 24. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 24. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 24.

In some embodiments, the ligand that binds to a CD3 is L2K. In someembodiments, the L2K ligand binds CD3. In some embodiments, the L2Kligand selectively binds CD3. In some embodiments, the L2K ligandspecifically binds CD3. In some embodiments, the L2K ligand binds CD3ε.In some embodiments, the L2K ligand selectively binds CD3ε. In someembodiments, the L2K ligand specifically binds CD3ε. In someembodiments, the murine L2K ligand is encoded by SEQ ID NO 25. In someembodiments, the L2K ligand comprises SEQ ID NO 26.

In some embodiments, the second polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 25. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 25. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 25. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 25. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 25. Insome embodiments, the second polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 25. Insome embodiments, the second polynucleotide comprises a nucleotidesequence of SEQ ID NO: 25.

In some embodiments, the ligand that binds a TCR complex comprises anamino acid sequence having at least 70% sequence identity with SEQ IDNO: 26. In some embodiments, the ligand that binds a TCR complexcomprises an amino acid sequence having at least 75% sequence identitywith SEQ ID NO: 26. In some embodiments, the ligand that binds a TCRcomplex comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 26. In some embodiments, the ligand that bindsa TCR complex comprises an amino acid sequence having at least 85%sequence identity with SEQ ID NO: 26. In some embodiments, the ligandthat binds a TCR complex comprises an amino acid sequence having atleast 90% sequence identity with SEQ ID NO: 26. In some embodiments, theligand that binds a TCR complex comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 26. In some embodiments,the ligand that binds a TCR complex comprises an amino acid sequence ofSEQ ID NO: 26.

Transmembrane domain and Cytosolic Domain

In some embodiments, a T cell antigen coupler includes a T cell receptorsignaling domain polypeptide. In some embodiments, the TCR signalingdomain polypeptide comprises a transmembrane domain. In someembodiments, the TCR signaling domain polypeptide comprises a cytosolicdomain. In some embodiments, the TCR signaling domain polypeptidecomprises a transmembrane domain and a cytosolic domain. In someembodiments, the cytosolic domain and transmembrane domains areoptionally joined by a linker. In some embodiments, the T cell receptorsignaling domain polypeptide comprises a TCR co-receptor domain. In someembodiments, the T cell receptor signaling domain polypeptide does notcomprise a TCR co-stimulator domain. In some embodiments, the TCRsignaling domain polypeptide comprises a transmembrane domain and/or acytosolic domain of a TCR co-receptor. In some embodiments, the TCRco-receptor is CD4, CD8, LAG3, or a chimeric variation thereof.

In some embodiments, the TCR co-receptor is CD4. In some embodiments,the TCR signaling domain polypeptide comprises the transmembrane andcytosolic domains of the CD4 co-receptor encoded by SEQ ID NO: 17. Insome embodiments, the TCR signaling domain polypeptide comprises thetransmembrane and cytosolic domains of the CD4 co-receptor comprisingSEQ ID NO: 18.

In some embodiments, the third polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 17. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 17. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 17. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 17. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 17. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 17. Insome embodiments, the third polynucleotide comprises a nucleotidesequence of SEQ ID NO: 17.

In some embodiments, the transmembrane domain and cytosolic domaincomprise an amino acid sequence having at least 70% sequence identitywith SEQ ID NO: 18. In some embodiments, the transmembrane domain andcytosolic domain comprise an amino acid sequence having at least 75%sequence identity with SEQ ID NO: 18. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 18. Insome embodiments, the transmembrane domain and cytosolic domain comprisean amino acid sequence having at least 85% sequence identity with SEQ IDNO: 18. In some embodiments, the transmembrane domain and cytosolicdomain comprise an amino acid sequence having at least 90% sequenceidentity with SEQ ID NO: 18. In some embodiments, the transmembranedomain and cytosolic domain comprise an amino acid sequence having atleast 95% sequence identity with SEQ ID NO: 18. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence of SEQ ID NO: 18.

In some embodiments, the TCR co-receptor is CD8. In some embodiments,the TCR co-receptor is CD8α. In some embodiments, the TCR signalingdomain polypeptide comprises the transmembrane and cytosolic domains ofthe CD8α co-receptor encoded by SEQ ID NO: 37. In some embodiments, theTCR signaling domain polypeptide comprises the transmembrane andcytosolic domains of the CD8α co-receptor comprising SEQ ID NO: 38.

In some embodiments, the third polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 37. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 37. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 37. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 37. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 37. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 37. Insome embodiments, the third polynucleotide comprises a nucleotidesequence of SEQ ID NO: 37.

In some embodiments, the transmembrane domain and cytosolic domaincomprise an amino acid sequence having at least 70% sequence identitywith SEQ ID NO: 38. In some embodiments, the transmembrane domain andcytosolic domain comprise an amino acid sequence having at least 75%sequence identity with SEQ ID NO: 38. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 38. Insome embodiments, the transmembrane domain and cytosolic domain comprisean amino acid sequence having at least 85% sequence identity with SEQ IDNO: 38. In some embodiments, the transmembrane domain and cytosolicdomain comprise an amino acid sequence having at least 90% sequenceidentity with SEQ ID NO: 38. In some embodiments, the transmembranedomain and cytosolic domain comprise an amino acid sequence having atleast 95% sequence identity with SEQ ID NO: 38. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence of SEQ ID NO: 38.

In some embodiments, the TCR signaling domain polypeptide comprises achimera of sequences or domains from co-receptors. In some embodiments,the TCR signaling domain polypeptide comprises a chimera of CD8α andCD8β, wherein the CD8α arginine rich region is replaced with the CD8βarginine rich region. In some embodiments, the T cell receptor signalingdomain polypeptide comprises the transmembrane and cytosolic domains ofthe CD8α+R(β) co-receptor chimera encoded by SEQ ID NO: 39. In someembodiments, the T cell receptor signaling domain polypeptide comprisesthe transmembrane and cytosolic domains of the CD8α+R(β) co-receptorchimera provided by SEQ ID NO: 40. In some embodiments, the TCRsignaling domain polypeptide comprises a chimera of CD8α and CD8β, theCD8α CXCP domain, which contains an Lck binding motif, is appended tothe C-terminus of the CD8β cytosolic domain. In some embodiments, the Tcell receptor signaling domain polypeptide comprises the transmembraneand cytosolic domains of the CD8β+Lck co-receptor chimera encoded by SEQID NO: 41. In some embodiments, the T cell receptor signaling domainpolypeptide comprises the transmembrane and cytosolic domains of theCD8β+Lck co-receptor chimera provided by SEQ ID NO: 42.

In some embodiments, the third polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 39. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 39. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 39. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 39. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 39. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 39. Insome embodiments, the third polynucleotide comprises a nucleotidesequence of SEQ ID NO: 39.

In some embodiments, the transmembrane domain and cytosolic domaincomprise an amino acid sequence having at least 70% sequence identitywith SEQ ID NO: 40. In some embodiments, the transmembrane domain andcytosolic domain comprise an amino acid sequence having at least 75%sequence identity with SEQ ID NO: 40. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 40. Insome embodiments, the transmembrane domain and cytosolic domain comprisean amino acid sequence having at least 85% sequence identity with SEQ IDNO: 40. In some embodiments, the transmembrane domain and cytosolicdomain comprise an amino acid sequence having at least 90% sequenceidentity with SEQ ID NO: 40. In some embodiments, the transmembranedomain and cytosolic domain comprise an amino acid sequence having atleast 95% sequence identity with SEQ ID NO: 40. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence of SEQ ID NO: 40.

In some embodiments, the third polynucleotide comprises a nucleotidesequence having at least 70% sequence identity with SEQ ID NO: 41. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 75% sequence identity with SEQ ID NO: 41. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 80% sequence identity with SEQ ID NO: 41. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 41. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 90% sequence identity with SEQ ID NO: 41. Insome embodiments, the third polynucleotide comprises a nucleotidesequence having at least 95% sequence identity with SEQ ID NO: 41. Insome embodiments, the third polynucleotide comprises a nucleotidesequence of SEQ ID NO: 41.

In some embodiments, the transmembrane domain and cytosolic domaincomprise an amino acid sequence having at least 70% sequence identitywith SEQ ID NO: 42. In some embodiments, the transmembrane domain andcytosolic domain comprise an amino acid sequence having at least 75%sequence identity with SEQ ID NO: 42. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence having at least 80% sequence identity with SEQ ID NO: 42. Insome embodiments, the transmembrane domain and cytosolic domain comprisean amino acid sequence having at least 85% sequence identity with SEQ IDNO: 42. In some embodiments, the transmembrane domain and cytosolicdomain comprise an amino acid sequence having at least 90% sequenceidentity with SEQ ID NO: 42. In some embodiments, the transmembranedomain and cytosolic domain comprise an amino acid sequence having atleast 95% sequence identity with SEQ ID NO: 42. In some embodiments, thetransmembrane domain and cytosolic domain comprise an amino acidsequence of SEQ ID NO: 42.

In some embodiments, the T cell receptor signaling domain polypeptidecomprises a TCR co-stimulator domain. In some embodiments, the TCRco-stimulator is ICOS. In some embodiments, the TCR co-stimulator isCD27. In some embodiments, the TCR co-stimulator is CD28. In someembodiments, the TCR co-stimulator is 4-1BB (CD137). In someembodiments, the TCR co-stimulator is OX40 (CD134). In some embodiments,the TCR co-stimulator is CD30. In some embodiments, the TCRco-stimulator is CD40. In some embodiments, the TCR co-stimulator islymphocyte fiction-associated antigen 1 (LFA-1). In some embodiments,the TCR co-stimulator is CD2. In some embodiments, the TCR co-stimulatoris CD7. In some embodiments, the TCR co-stimulator is LIGHT. In someembodiments, the TCR co-stimulator is NKG2C. In some embodiments, theTCR co-stimulator is B7-H3. In some embodiments, the TCR co-stimulatoris a ligand that specifically binds CD83.

In some embodiments, the TCR signaling domain polypeptide comprises atransmembrane domain and/or a cytosolic domain of a TCR co-inhibitor. Insome embodiments, the TCR co-inhibitor is PD-1. In some embodiments, theTCR co-inhibitor is TIM3. In some embodiments, the TCR co-inhibitor isLAG-3. In some embodiments, the TCR co-inhibitor is TIGIT. In someembodiments, the TCR co-inhibitor is BTLA. In some embodiments, the TCRco-inhibitor is CD160. In some embodiments, the TCR co-inhibitor isCD37.

In some embodiments, the TCR signaling domain polypeptide includes botha cytosolic domain and a transmembrane domain of a TCR co-receptor orco-stimulator protein. In some embodiments, the cytosolic domain andtransmembrane domain are from the same co-receptor or co-stimulator orfrom different co-receptors or co-stimulators. In some embodiments, theTAC further comprises other polypeptides that directly or indirectly actto target or activate the T cell.

Linkers, Connectors, and Configurations

In some embodiments, a nucleic acid disclosed herein is in an order of(1) a first polynucleotide encoding a target-specific ligand; (2) asecond polynucleotide encoding a ligand that binds a TCR complex; (3) athird polynucleotide encoding a transmembrane domain and a cytosolicdomain. In some embodiments, a nucleic acid disclosed herein is in anorder of (1) a first polynucleotide encoding a target-specific ligand;(2) a second polynucleotide encoding a ligand that binds a TCR complex;(3) a third polynucleotide encoding a transmembrane domain and acytosolic domain, wherein the order is 5′ end to 3′ end. In someembodiments, a nucleic acid disclosed herein is in an order of (1) afirst polynucleotide encoding a target-specific ligand; (2) a secondpolynucleotide encoding a ligand that binds a TCR complex; (3) a thirdpolynucleotide encoding a transmembrane domain and a cytosolic domain,wherein the order is 3′ end to 5′ end. In some embodiments, a nucleicacid described herein is in an order of (1) a first polynucleotideencoding a ligand that binds a TCR complex; (2) a second polynucleotideencoding a target-specific ligand; (3) a third polynucleotide encoding atransmembrane domain and a cytosolic domain. In some embodiments, anucleic acid described herein is in an order of (1) a firstpolynucleotide encoding a ligand that binds a TCR complex; (2) a secondpolynucleotide encoding a target-specific ligand; (3) a thirdpolynucleotide encoding a transmembrane domain and a cytosolic domain,wherein the order is 5′ end to 3′ end. In some embodiments, a nucleicacid described herein is in an order of (1) a first polynucleotideencoding a ligand that binds a TCR complex; (2) a second polynucleotideencoding a target-specific ligand; (3) a third polynucleotide encoding atransmembrane domain and a cytosolic domain, wherein the order is 3′ endto 5′ end.

In some embodiments, the first nucleic acid encodes a first polypeptide,the second nucleic acid encodes a second polypeptide, and the thirdnucleic acid encodes a third polypeptide. In some embodiments, the firstpolypeptide, the second polypeptide, and the third polypeptide aredirectly fused. For example, the target-specific ligand and the T cellreceptor signaling domain polypeptide are both fused to the ligand thatbinds the TCR complex. In some embodiments, the first polypeptide, thesecond polypeptide, and the third polypeptide are joined by at least onelinker. In some embodiments, the first polypeptide and the secondpolypeptide are directly fused, and joined to the third polypeptide by alinker. In some embodiments, the second polypeptide and the thirdpolypeptide are directly fused, and joined to the first polypeptide by alinker.

In some embodiments, the linker is a peptide linker. In someembodiments, the peptide linker comprises 1 to 40 amino acids. In someembodiments, the peptide linker comprises 1 to 30 amino acids. In someembodiments, the peptide linker comprises 1 to 15 amino acids. In someembodiments, the peptide linker comprises 1 to 10 amino acids. In someembodiments, the peptide linker comprises 1 to 6 amino acids. In someembodiments, the peptide linker comprises 30 to 40 amino acids. In someembodiments, the peptide linker comprises 32 to 36 amino acids. In someembodiments, the peptide linker comprises 5 to 30 amino acids. In someembodiments, the peptide linker comprises 5 amino acids. In someembodiments, the peptide linker comprises 10 amino acids. In someembodiments, the peptide linker comprises 15 amino acids. In someembodiments, the peptide linker comprises 20 amino acids. In someembodiments, the peptide linker comprises 25 amino acids. In someembodiments, the peptide linker comprises 30 amino acids.

In some embodiments, the peptide linker comprises a G₄S₃ linker (SEQ IDNO: 74). In some embodiments, the peptide linker comprises SEQ ID NOs:11, 12, 15, 16, 19, 20, or variants or fragments thereof.

In some embodiments, the peptide linker that joins the target-specificligand to the ligand that binds a TCR complex (e.g. UCHT1) is known asthe connector to distinguish this protein domain from other linkers inthe Tri-TAC. The connector is of any size. In some embodiments, theconnector between ligand that binds a TCR complex and a target-specificligand is a short helix comprising SEQ ID NO ID: 28. In someembodiments, the connector between ligand that binds a TCR complex and atarget-specific ligand is a short helix encoded by SEQ ID NO ID: 27. Insome embodiments, the connector between ligand that binds a TCR complexand a target-specific ligand is a long helix comprising SEQ ID NO ID:30. In some embodiments, the connector between ligand that binds a TCRcomplex and a target-specific ligand is a long helix encoded by SEQ IDNO ID: 29. In some embodiments, the connector between ligand that bindsa TCR complex and a target-specific ligand is a large domain comprisingSEQ ID NO ID: 32. In some embodiments, the connector between ligand thatbinds a TCR complex and a target-specific ligand is a large domainencoded by SEQ ID NO ID: 31.

In some embodiments, a nucleic acid disclosed herein comprises a leadersequence. In some embodiments, the leader sequence comprises anucleotide sequence having at least 70% sequence identity with SEQ IDNO: 5, 47, or 49. In some embodiments, the leader sequence comprises anucleotide sequence having at least 75% sequence identity with SEQ IDNO: 5, 47, or 49. In some embodiments, the leader sequence comprises anucleotide sequence having at least 80% sequence identity with SEQ IDNO: 5, 47, or 49. In some embodiments, the leader sequence comprises anucleotide sequence having at least 85% sequence identity with SEQ IDNO: 5, 47, or 49. In some embodiments, the leader sequence comprises anucleotide sequence having at least 90% sequence identity with SEQ IDNO: 5, 47, or 49. In some embodiments, the leader sequence comprises anucleotide sequence having at least 95% sequence identity with SEQ IDNO: 5, 47, or 49. In some embodiments, the leader sequence comprises anucleotide sequence of SEQ ID NO: 5, 47, or 49.

In some embodiments, a nucleic acid disclosed herein comprises a leadersequence. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 70% sequence identity with SEQ ID NO: 6,48, or 50. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 75% sequence identity with SEQ ID NO: 6,48, or 50. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 80% sequence identity with SEQ ID NO: 6,48, or 50. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 85% sequence identity with SEQ ID NO: 6,48, or 50. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 90% sequence identity with SEQ ID NO: 6,48, or 50. In some embodiments, the leader sequence comprises an aminoacid sequence having at least 95% sequence identity with SEQ ID NO: 6,48, or 50. In some embodiments, the leader sequence comprises an aminoacid sequence of SEQ ID NO: 6, 48, or 50.

The Tri-TAC is contemplated to be present in various configurations andcombinations of (a) target-specific ligand, (b) a ligand that binds aTCR complex, and (c) a TCR signaling domain, as disclosed herein.

In some embodiments, the Tri-TAC comprises (a) a target-specific ligand,(b) a single-chain antibody (scFv) that binds CD3c, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a target-specific ligand, (b)UCHT1, and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) atarget-specific ligand, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a target-specific ligand, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a target-specific ligand, (b)huUCHT1 (Y177T), and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) atarget-specific ligand, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD4 co-receptor. In some embodiments, the Tri-TACcomprises (a) a target-specific ligand, (b) F6A, and (c) a transmembraneand cytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a target-specific ligand, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a DARPin, (b) UCHT1, and(c) a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) UCHT1 (Y182T), and(c) a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) huUCHT1 (Y177T),and (c) a transmembrane and cytosolic domain of the CD4 co-receptor. Insome embodiments, the Tri-TAC comprises (a) a DARPin, (b) OKT3, and (c)a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) F6A, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a scFv, (b) UCHT1, and(c) a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) UCHT1 (Y182T), and(c) a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) huUCHT1 (Y177T), and(c) a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) OKT3, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) F6A, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a HER-2-specific DARPin,(b) UCHT1, and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) aHER-2-specific DARPin, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a HER-2-specific DARPin, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a target-specific ligand, (b)huUCHT1 (Y177T), and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) aHER-2-specific DARPin, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD4 co-receptor. In some embodiments, the Tri-TACcomprises (a) a HER-2-specific DARPin, (b) F6A, and (c) a transmembraneand cytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a HER-2-specific DARPin, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b)UCHT1, and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) aBCMA-specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT1(Y177T), and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) aBCMA-specific ScFv, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD4 co-receptor. In some embodiments, the Tri-TACcomprises (a) a BCMA-specific ScFv, (b) F6A, and (c) a transmembrane andcytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a BCMA-specific ScFv, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a CD19-specific ScFv, (b)UCHT1, and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) aCD19-specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a CD19-specific ScFv, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a CD19-specific ScFv, (b) huUCHT1(Y177T), and (c) a transmembrane and cytosolic domain of the CD4co-receptor. In some embodiments, the Tri-TAC comprises (a) aCD19-specific ScFv, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD4 co-receptor. In some embodiments, the Tri-TACcomprises (a) a CD19-specific ScFv, (b) F6A, and (c) a transmembrane andcytosolic domain of the CD4 co-receptor. In some embodiments, theTri-TAC comprises (a) a CD19-specific ScFv, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD4 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a target-specific ligand,(b) a single-chain antibody (scFv) that binds CD3c, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a target-specific ligand, (b)UCHT1, and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) atarget-specific ligand, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a target-specific ligand, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a target-specific ligand, (b)huUCHT1 (Y177T), and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) atarget-specific ligand, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD8 co-receptor. In some embodiments, the Tri-TACcomprises (a) a target-specific ligand, (b) F6A, and (c) a transmembraneand cytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a target-specific ligand, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a DARPin, (b) UCHT1, and(c) a transmembrane and cytosolic domain of the CD4 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) UCHT1 (Y182T), and(c) a transmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) huUCHT1 (Y177T),and (c) a transmembrane and cytosolic domain of the CD8 co-receptor. Insome embodiments, the Tri-TAC comprises (a) a DARPin, (b) OKT3, and (c)a transmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) F6A, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a DARPin, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a scFv, (b) UCHT1, and(c) a transmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) UCHT1 (Y182T), and(c) a transmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) huUCHT1 (Y177T), and(c) a transmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) OKT3, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) F6A, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a scFv, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a HER-2-specific DARPin,(b) UCHT1, and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) aHER-2-specific DARPin, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a HER-2-specific DARPin, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a target-specific ligand, (b)huUCHT1 (Y177T), and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) aHER-2-specific DARPin, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD8 co-receptor. In some embodiments, the Tri-TACcomprises (a) a HER-2-specific DARPin, (b) F6A, and (c) a transmembraneand cytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a HER-2-specific DARPin, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b)UCHT1, and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) aBCMA-specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a BCMA-specific ScFv, (b) huUCHT1(Y177T), and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) aBCMA-specific ScFv, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD8 co-receptor. In some embodiments, the Tri-TACcomprises (a) a BCMA-specific ScFv, (b) F6A, and (c) a transmembrane andcytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a BCMA-specific ScFv, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor.

In some embodiments, the Tri-TAC comprises (a) a CD19-specific ScFv, (b)UCHT1, and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) aCD19-specific ScFv, (b) UCHT1 (Y182T), and (c) a transmembrane andcytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a CD19-specific ScFv, (b) huUCHT1, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor. In someembodiments, the Tri-TAC comprises (a) a CD19-specific ScFv, (b) huUCHT1(Y177T), and (c) a transmembrane and cytosolic domain of the CD8co-receptor. In some embodiments, the Tri-TAC comprises (a) aCD19-specific ScFv, (b) OKT3, and (c) a transmembrane and cytosolicdomain of the CD8 co-receptor. In some embodiments, the Tri-TACcomprises (a) a CD19-specific ScFv, (b) F6A, and (c) a transmembrane andcytosolic domain of the CD8 co-receptor. In some embodiments, theTri-TAC comprises (a) a CD19-specific ScFv, (b) L2K, and (c) atransmembrane and cytosolic domain of the CD8 co-receptor.

In some embodiments, the Tri-TAC draws CD3 and TCR into lipid raftregions of the membrane, and brings Lck into the proximity of the TCR,similar to natural MHC binding.

In some embodiments, the TAC disclosed herein is the anti-HER-2 DARPinTri-TAC (also referred to as configuration 1; SEQ ID NO: 1 and 2)includes, in order:

i) the anti-HER-2 Tri-TAC leader sequence (secretion signal) (SEQ ID NO:5 and 6)

ii) DARPin specific for HER-2 antigen (SEQ ID NO: 7 and 8)

iii) Myc tag (SEQ ID NO: 9 and 10)

iv) Connector (SEQ ID NO: 11 and 12)

v) UCHT1 (SEQ ID NO: 13 and 14)

vi) Linker (SEQ ID NO: 15 and 16)

vii) CD4 (SEQ ID NO: 17 and 18).

In some embodiments, the TAC disclosed herein is a HER2-TAC. In someembodiments, the HER2-TAC comprises a nucleotide sequence having atleast 70% sequence identity with SEQ ID NO: 65. In some embodiments, theHER2-TAC comprises a nucleotide sequence having at least 75% sequenceidentity with SEQ ID NO: 65. In some embodiments, the HER2-TAC comprisesa nucleotide sequence having at least 80% sequence identity with SEQ IDNO: 65. In some embodiments, the HER2-TAC comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 65. Insome embodiments, the HER2-TAC comprises a nucleotide sequence having atleast 90% sequence identity with SEQ ID NO: 65. In some embodiments, theHER2-TAC comprises a nucleotide sequence having at least 95% sequenceidentity with SEQ ID NO: 65. In some embodiments, the HER2-TAC comprisesa nucleotide sequence of SEQ ID NO: 65.

In some embodiments, the HER2-TAC comprises an amino acid sequencehaving at least 70% sequence identity with SEQ ID NO: 66. In someembodiments, the HER2-TAC comprises an amino acid sequence having atleast 75% sequence identity with SEQ ID NO: 66. In some embodiments, theHER2-TAC comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 66. In some embodiments, the HER2-TAC comprisesan amino acid sequence having at least 85% sequence identity with SEQ IDNO: 66. In some embodiments, the HER2-TAC an amino acid sequence havingat least 90% sequence identity with SEQ ID NO: 66. In some embodiments,the HER2-TAC comprises an amino acid sequence having at least 95%sequence identity with SEQ ID NO: 66. In some embodiments, the HER2-TACcomprises an amino acid sequence of SEQ ID NO: 66.

In some embodiments, the TAC disclosed herein is a HER2-TAC. In someembodiments, the HER2-TAC comprises a nucleotide sequence having atleast 70% sequence identity with SEQ ID NO: 67. In some embodiments, theHER2-TAC comprises a nucleotide sequence having at least 75% sequenceidentity with SEQ ID NO: 67. In some embodiments, the HER2-TAC comprisesa nucleotide sequence having at least 80% sequence identity with SEQ IDNO: 67. In some embodiments, the HER2-TAC comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 67. Insome embodiments, the HER2-TAC comprises a nucleotide sequence having atleast 90% sequence identity with SEQ ID NO: 67. In some embodiments, theHER2-TAC comprises a nucleotide sequence having at least 95% sequenceidentity with SEQ ID NO: 67. In some embodiments, the HER2-TAC comprisesa nucleotide sequence of SEQ ID NO: 67.

In some embodiments, the HER2-TAC comprises an amino acid sequencehaving at least 70% sequence identity with SEQ ID NO: 68. In someembodiments, the HER2-TAC comprises an amino acid sequence having atleast 75% sequence identity with SEQ ID NO: 68. In some embodiments, theHER2-TAC comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 68. In some embodiments, the HER2-TAC comprisesan amino acid sequence having at least 85% sequence identity with SEQ IDNO: 68. In some embodiments, the HER2-TAC an amino acid sequence havingat least 90% sequence identity with SEQ ID NO: 68. In some embodiments,the HER2-TAC comprises an amino acid sequence having at least 95%sequence identity with SEQ ID NO: 68. In some embodiments, the HER2-TACcomprises an amino acid sequence of SEQ ID NO: 68.

In some embodiments, the TAC disclosed herein is a BCMA-TAC. In someembodiments, the BCMA-TAC comprises a nucleotide sequence having atleast 70% sequence identity with SEQ ID NO: 55, 57, 59 or 61. In someembodiments, the BCMA-TAC comprises a nucleotide sequence having atleast 75% sequence identity with SEQ ID NO: 55, 57, 59 or 61. In someembodiments, the BCMA-TAC comprises a nucleotide sequence having atleast 80% sequence identity with SEQ ID NO: 55, 57, 59 or 61. In someembodiments, the BCMA-TAC comprises a nucleotide sequence having atleast 85% sequence identity with SEQ ID NO: 55, 57, 59 or 61. In someembodiments, the BCMA-TAC comprises a nucleotide sequence having atleast 90% sequence identity with SEQ ID NO: 55, 57, 59 or 61. In someembodiments, the BCMA-TAC comprises a nucleotide sequence having atleast 95% sequence identity with SEQ ID NO: 55, 57, 59 or 61. In someembodiments, the BCMA-TAC comprises a nucleotide sequence of SEQ ID NO:55, 57, 59 or 61.

In some embodiments, the BCMA-TAC comprises an amino acid sequencehaving at least 70% sequence identity with SEQ ID NO: 56, 58, 60 or 62.In some embodiments, the BCMA-TAC comprises an amino acid sequencehaving at least 75% sequence identity with SEQ ID NO: 56, 58, 60 or 62.In some embodiments, the BCMA-TAC comprises an amino acid sequencehaving at least 80% sequence identity with SEQ ID NO: 56, 58, 60 or 62.In some embodiments, the BCMA-TAC comprises an amino acid sequencehaving at least 85% sequence identity with SEQ ID NO: 56, 58, 60 or 62.In some embodiments, the BCMA-TAC comprises an amino acid sequencehaving at least 90% sequence identity with SEQ ID NO: 56, 58, 60 or 62.In some embodiments, the BCMA-TAC comprises an amino acid sequencehaving at least 95% sequence identity with SEQ ID NO: 56, 58, 60 or 62.In some embodiments, the BCMA-TAC comprises an amino acid sequence ofSEQ ID NO: 56, 58, 60 or 62.

In some embodiments, the TAC disclosed herein is a CD19-TAC. In someembodiments, the CD19-TAC comprises a nucleotide sequence having atleast 70% sequence identity with SEQ ID NO: 63. In some embodiments, theCD19-TAC comprises a nucleotide sequence having at least 75% sequenceidentity with SEQ ID NO: 63. In some embodiments, the CD19-TAC comprisesa nucleotide sequence having at least 80% sequence identity with SEQ IDNO: 63. In some embodiments, the CD19-TAC comprises a nucleotidesequence having at least 85% sequence identity with SEQ ID NO: 63. Insome embodiments, the CD19-TAC comprises a nucleotide sequence having atleast 90% sequence identity with SEQ ID NO: 63. In some embodiments, theCD19-TAC comprises a nucleotide sequence having at least 95% sequenceidentity with SEQ ID NO: 63. In some embodiments, the CD19-TAC comprisesa nucleotide sequence of SEQ ID NO: 63.

In some embodiments, the CD19-TAC comprises an amino acid sequencehaving at least 70% sequence identity with SEQ ID NO: 64. In someembodiments, the CD19-TAC comprises an amino acid sequence having atleast 75% sequence identity with SEQ ID NO: 64. In some embodiments, theCD19-TAC comprises an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO: 64. In some embodiments, the CD19-TAC comprisesan amino acid sequence having at least 85% sequence identity with SEQ IDNO: 64. In some embodiments, the CD19-TAC comprises an amino acidsequence having at least 90% sequence identity with SEQ ID NO: 64. Insome embodiments, the CD19-TAC comprises an amino acid sequence havingat least 95% sequence identity with SEQ ID NO: 64. In some embodiments,the CD19-TAC comprises an amino acid sequence of SEQ ID NO: 64.

Polypeptides and Vector Constructs

Disclosed herein, in certain embodiments, are polypeptides encoded bythe nucleic acid sequence as disclosed herein. Also disclosed herein,are vectors comprising the nucleic acid sequence as disclosed herein. Insome embodiments, the vectors further comprise a promoter. In someembodiments, the promoter is functional in a mammalian cell. Promoters,regions of DNA that initiate transcription of a particular nucleic acidsequence, are well known in the art. A “promoter functional in amammalian cell” refers to a promoter that drives expression of theassociated nucleic acid sequence in a mammalian cell. A promoter thatdrives expression of a nucleic acid sequence is referred to as being“operably connected” to the nucleic acid sequence.

A variety of delivery vectors and expression vehicles are employed tointroduce nucleic acids described herein into a cell.

Disclosed herein, in certain embodiments, are polynucleotides comprisedin a vector to provide a vector construct, also herein referred to as avector. In some embodiments, the present disclosure provides a vectorcomprising:

-   -   a. a first polynucleotide encoding a target-specific ligand;    -   b. a second polynucleotide encoding a ligand that binds a        protein associated with a TCR complex;    -   c. a third polynucleotide encoding a T cell receptor signaling        domain polypeptide; and    -   d. a promoter that is functional in a mammalian cell.

In some embodiments, the target of the target-specific ligand binds toHER-2, BCMA, or CD19. In some embodiments, the target-specific ligand isa DARPin that selectively binds a HER-2 (erbB-2) antigen. In someembodiments, the target-specific ligand is a DARPin that specificallybinds a HER-2 (erbB-2) antigen. In some embodiments, the DARPin targetedto HER-2 (erb-2) comprises SEQ ID NO: 7 or SEQ ID NO: 8. In someembodiments, the target-specific ligand is a scFv that selectively bindsBCMA. In some embodiments, the target-specific ligand is a scFv thatspecifically binds BCMA. In some embodiments, the scFv that binds BCMAcomprises SEQ ID NO: 33 or SEQ ID NO: 34. In some embodiments, thetarget-specific ligand is a scFv that selectively binds CD19. In someembodiments, the target-specific ligand is a scFv that specificallybinds CD19. In some embodiments, the scFv that binds CD19 comprises SEQID NO: 35 or SEQ ID NO: 36.

In some embodiments, the ligand that binds a protein associated with aTCR complex is UCHT1, humanized UCHT1 (huUCHT1), OKT3, F6A, or L2K. Insome embodiments, the ligand that binds a protein associated with a TCRcomplex is UCHT1, or a variant thereof. In some embodiments, the ligandthat binds a protein associated with a TCR complex is UCHT1 and isencoded by SEQ ID NO: 13. In some embodiments, the ligand that binds aprotein associated with a TCR complex is UCHT1 and comprises SEQ ID NO:14 In some embodiments, the UCHT1 ligand that binds a protein associatedwith a TCR complex has a Y182T mutation (UCHT1 (Y182T)) and is encodedby SEQ ID NO: 71. In some embodiments, the ligand that binds a proteinassociated with a TCR complex is UCHT1 (Y182T) and comprises SEQ ID NO:72. In some embodiments, the ligand that binds a protein associated witha TCR complex is humanized UCHT1 (huUCHT1), or a variant thereof. Insome embodiments, the ligand that binds a protein associated with a TCRcomplex is humanized UCHT1 (huUCHT1) and is encoded by SEQ ID NO: 43. Insome embodiments, the ligand that binds a protein associated with a TCRcomplex is huUCHT1 and comprises SEQ ID NO: 44. In some embodiments, thehuUCHT1 ligand that binds a protein associated with a TCR complex has aY177T mutation (huUCHT1 (Y177T)) and is encoded by SEQ ID NO: 45. Insome embodiments, the ligand that binds a protein associated with a TCRcomplex is huUCHT1 (Y177T) and comprises SEQ ID NO: 46.

In some embodiments, the ligand that binds a protein associated with aTCR complex is OKT3, or a variant thereof. In some embodiments, theligand that binds a protein associated with a TCR complex is OKT3 and isencoded by SEQ ID NO: 21. In some embodiments, the ligand that binds aprotein associated with a TCR complex is OKT3 and comprises SEQ ID NO:22.

In some embodiments, the ligand that binds a protein associated with aTCR complex is F6A, or a variant thereof. In some embodiments, theligand that binds a protein associated with a TCR complex is F6A and isencoded by SEQ ID NO: 23. In some embodiments, the ligand that binds aprotein associated with a TCR complex is F6A and comprises SEQ ID NO:24.

In some embodiments, the ligand that binds a protein associated with aTCR complex is L2K, or a variant thereof. In some embodiments, theligand that binds a protein associated with a TCR complex is L2K and isencoded by SEQ ID NO: 25. In some embodiments, the ligand that binds aprotein associated with a TCR complex is L2K and comprises SEQ ID NO:26.

In some embodiments, the protein associated with a TCR complex is CD3.In some embodiments, the protein associated with a TCR complex is CD3ε.

In some embodiments, the TCR signaling domain polypeptide comprises atransmembrane domain and/or a cytosolic domain of a TCR co-receptor. Insome embodiments, the TCR co-receptor is CD4, CD8, LAG3, or a chimericvariation thereof.

In some embodiments, the first polynucleotide and third polynucleotideare fused to the second polynucleotide and the coding sequence isoperably connected to the promoter. In some embodiments, the secondpolynucleotide and third polynucleotide are fused to the firstpolynucleotide and the coding sequence is operably connected to thepromoter. In some embodiments, the vector is designed for expression inmammalian cells such as T cells. In some embodiments, the vector is aviral vector. In some embodiments, the viral vector is a retroviralvector.

In some embodiments, vectors that are useful comprise vectors derivedfrom lentiviruses, Murine Stem Cell Viruses (MSCV), pox viruses,oncoretroviruses, adenoviruses, and adeno-associated viruses. Otherdelivery vectors that are useful comprise vectors derived from herpessimplex viruses, transposons, vaccinia viruses, human papilloma virus,Simian immunodeficiency viruses, HTLV, human foamy virus and variantsthereof. Further vectors that are useful comprise vectors derived fromspumaviruses, mammalian type B retroviruses, mammalian type Cretroviruses, avian type C retroviruses, mammalian type D retrovirusesand HTLV/BLV type retroviruses. One example of a lentiviral vectoruseful in the disclosed compositions and methods is the pCCL4 vector.

In some embodiments, the nucleic acid is a recombinant, or engineered,nucleic acid. In some embodiments, the first, second and/or thirdpolynucleotides are recombinant, or engineered, polynucleotides. In someembodiments, the polynucleotides described herein are be modified ormutated to optimize the function of the encoded polypeptide and/or thefunction, activity and/or expression of the T cell antigen coupler. Insome embodiments, the nucleic acid encodes a polypeptide.

In some embodiments, modifications are made to the polynucleotidesequences including vector sequences and polypeptides sequencesdisclosed herein. Modifications include substitution, insertion ordeletion of nucleotides or amino acids or altering the relativepositions or order of nucleotides or amino acids.

Expression in T Cells

Disclosed herein, in certain embodiments, are engineered T cellscomprising the nucleic acid sequences disclosed herein, or the vectorsdisclosed herein. Disclosed herein, in certain embodiments, are human Tcells engineered to express a Tri-TAC disclosed herein. In someembodiments, the T cell expresses a Tri-TAC disclosed herein. Furtherdisclosed herein, are T cells transduced or transfected with T cellantigen coupler or a vector comprising a Tri-TAC. In some embodiments,the T cell is an isolated T cell.

In some embodiments, the human T cells engineered to express a Tri-TACdemonstrate functionality equivalent to a conventional CAR in vitro. Insome embodiments, T cells engineered with the Tri-TAC demonstratefunctionality superior to a conventional CAR in vitro. Disclosed herein,in some embodiments, are human T cells engineered with a Tri-TAC thatdemonstrate enhanced safety compared to traditional CARs. In someembodiments, human T cells engineered to express a Tri-TAC demonstrateenhanced safety compared to traditional CARs.

T cells, in some embodiments, are obtained from a number of sources,including, but not limited to blood (for example, peripheral bloodmononuclear cells), bone marrow, thymus tissue, lymph node tissue, cordblood, thymus tissue, tissue from an infection site, spleen tissue, ortumors. In some embodiments, the T cells are autologous T cells. In someembodiments, the T cells are obtained from a cell line of T cells. Insome embodiments, the T cells are obtained from donors (allogeneic Tcells). In some embodiments, the T cells are obtained by differentiationof embryonic or adult stem cells or from induced pluripotent stem cells.In some embodiments, regardless of the source of T cells, the T cellshave been modified so that they lack expression of an endogenous TCRand/or permanently or transiently lack expression of MHC/HLA molecules(universal donor T cells). In some embodiments, the T cells areautologous with respect to the subject. In some embodiments, the cellsare allogeneic, syngeneic, or xenogeneic with respect to the subject.

In some embodiments, once obtained, the T cells are optionally enrichedin vitro. In some embodiments, a population of cells is enriched bypositive or negative selection. Further, the T cells are optionallyfrozen or cryopreserved and then thawed at a later date.

In some embodiments, T cells are activated and/or expanded before orafter introducing the Tri-TAC to the T cells. In some embodiments, the Tcells are expanded by contact with a surface having attached thereto anagent that stimulates a CD3/TCR complex associated signal and a ligandthat stimulates a co-stimulator molecule on the surface of the T cells.In some embodiments, the T cells are expanded by contact with one ormore soluble agents that stimulate CD3/TCR complex signaling andco-stimulator molecule signaling.

In some embodiments, the T cells are transduced or transfected withnucleic acid sequences. The transduced or transfected T cells expressproteins coded for by the transfected or transduced nucleic acidsequences. A nucleic acid may be introduced into a cell by physical,chemical, or biological means. Physical means include, but are notlimited to, microinjection, electroporation, particle bombardment,lipofection and calcium phosphate precipitation. Biological meansinclude the use of DNA and RNA vectors.

Viral vectors, including retroviral vectors, are used to introduce andexpress a nucleic acid into a T cell. Viral vectors include vectorsderived from lentivirus, Murine Stem Cell Viruses (MSCV), pox viruses,herpes simplex virus I, adenovirus and adeno-associated viruses. Thevector optionally includes a promoter that drives expression of thetransduced nucleic acid molecule in a T cell (e.g., a CMV promoter, eF1apromoter, or MSCV promoter).

Any suitable assay is used to confirm the presence and/or expression ofthe transduced nucleic acid sequence and/or the polypeptide encoded bythe nucleic acid in the T cell. Assays include, but are not limited to,Southern and Northern blotting, RT-PCR and PCR, ELISA, Western blotting,and flow cytometry.

A T cell expressing a TAC has increased T cell activation in thepresence of an antigen compared to a T cell not expressing a TAC and/oras compared to a T cell expressing a traditional CAR. Increased T cellactivation is ascertained by numerous methods, including but not limitedto, increased tumor cell line killing, increased cytokine production,increased cytolysis, increased degranulation and/or increased expressionof activation markers such as CD107α, IFNγ, IL2 or TNFα. In someembodiments, increases are measured in an individual cell or in apopulation of cells.

The terms “increased” or “increasing” as used herein refer to at least a1%, 2%, 5%, 10%, 25%, 50%, 100% or 200% increase in a T cell orpopulation of T cells expressing a TAC compared to a T cell orpopulation of T cells not expressing a TAC and/or as compared to a Tcell or population of T cells expressing a traditional CAR.

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising an engineered T cell disclosed herein(transduced with and/or expressing a TAC), and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers include, butare not limited to, buffers such as neutral buffered saline, phosphatebuffered saline and the like; carbohydrates such as glucose, mannose,sucrose or dextrans, mannitol; proteins; polypeptides or amino acidssuch as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); or preservatives. Insome embodiments, the engineered T cells are formulated for intravenousadministration.

Pharmaceutical compositions are administered in a manner appropriate tothe disease to be treated (or prevented). The quantity and frequency ofadministration is determined by such factors as the condition of thepatient, and the type and severity of the patient's disease, althoughappropriate dosages are determined by clinical trials. When “animmunologically effective amount,” “an anti-tumor effective amount,” “atumor-inhibiting effective amount,” or “therapeutic amount” isindicated, the precise amount of the compositions of the presentinvention to be administered is determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject).

In some embodiments, the modified T cells and/or pharmaceuticalcompositions described herein are administered at a dosage of 10¹ to10¹⁵ cells per kg body weight, 10⁴ to 10⁹ cells per kg body weight,optionally 10⁵ to 10⁸ cells per kg body weight, 10⁶ to 10⁷ cells per kgbody weight or 10⁵ to 10⁶ cells per kg body weight, including allinteger values within those ranges. In some embodiments, the modified Tcells and/or pharmaceutical compositions described herein areadministered at a dosage of greater than 10¹ cells per kg body weight.In some embodiments, the modified T cells and/or pharmaceuticalcompositions described herein are administered at a dosage of less than10¹⁵ cells per kg body weight.

In some embodiments, the modified T cells and/or pharmaceuticalcompositions described herein are administered at a dosage of 0.5×10⁶cells, 2×10⁶ cells, 4×10⁶ cells, 5×10⁶ cells, 1.2×10⁷ cells, 2×10⁷cells, 5×10⁷ cells, 2×10⁸ cells, 5×10⁸ cells, 2×10⁹ cells, 0.5-2000×10⁶cells, 0.5-2×10⁶ cells, 0.5-2×10⁷ cells, 0.5-2×10⁸ cells, or 0.5-2×10⁹cells, including all integer values within those ranges.

In some embodiments, T cell compositions are administered multiple timesat these dosages. In some embodiments, the dosage is administered asingle time or multiple times, for example daily, weekly, biweekly, ormonthly, hourly, or is administered upon recurrence, relapse orprogression of the cancer being treated. The cells, in some embodiments,are administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

The pharmaceutical composition is substantially free of, e.g., there areno detectable levels of a contaminant, e.g., selected from the groupconsisting of endotoxin, mycoplasma, replication competent lentivirus(RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD³/anti-CD28coated beads, mouse antibodies, pooled human serum, bovine serumalbumin, bovine serum, culture media components, vector packaging cellor plasmid components, a bacterium a fungus, mycoplasma, IL-2, and IL-7.

In some embodiments, engineered T-cells disclose herein are administeredto a subject and blood is subsequently redrawn (or apheresis performed),T-cells therefrom are activated and reinfused into the patient withengineered T cells. This process, in some embodiments, is carried outmultiple times every few weeks. T-cells are activated from blood drawsof from 10 cc to 400 cc. T-cells are activated from blood draws of 20cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The modified/engineered T cells and/or pharmaceutical compositions areadministered by methods including, but not limited to, aerosolinhalation, injection, infusion, ingestion, transfusion, implantation ortransplantation. The modified T cells and/or pharmaceutical compositionsare administered to a subject transarterially, subcutaneously,intradermally, intratumorally, intranodally, intrameduliary,intramuscularly, by intravenous (i.v.) injection, by intravenous (i.v.)infusion, or intraperitoneally. The modified/engineered T cells and/orpharmaceutical compositions thereof are administered to a patient byintradermal or subcutaneous injection. The modified/engineered T cellsand/or pharmaceutical compositions thereof are administered by i.v.injection. The modified/engineered T cells and/or pharmaceuticalcompositions thereof are injected directly into a tumor, lymph node, orsite of infection.

The modified/engineered T cells T cells and/or pharmaceuticalcompositions are administered in a volume of about 5 mL, 10 mL, 15 mL,20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400mL, or 500 mL.

The modified/engineered T cells T cells and/or pharmaceuticalcompositions are administered in a volume of at greater than at mostabout 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL,150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.

The modified/engineered T cells T cells and/or pharmaceuticalcompositions are administered in a volume of at least about 5 mL, 10 mL,15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300mL, 400 mL, or 500 mL.

A pharmaceutical composition is prepared by per se known methods for thepreparation of pharmaceutically acceptable compositions that areadministered to subjects, such that an effective quantity of the T cellsare combined in a mixture with a pharmaceutically acceptable carrier.Suitable carriers are described, for example, in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20^(th)ed., Mack Publishing Company, Easton, Pa., USA, 2000). On this basis,the compositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablecarriers or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

Suitable pharmaceutically acceptable carriers include essentiallychemically inert and nontoxic compositions that do not interfere withthe effectiveness of the biological activity of the pharmaceuticalcomposition. Examples of suitable pharmaceutical carriers include, butare not limited to, water, saline solutions, glycerol solutions,N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. In someembodiments, such compositions contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for direct administration to the patient.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions.

A pharmaceutical composition disclosed herein is formulated into avariety of forms and administered by a number of different means. Apharmaceutical formulation is administered orally, rectally, orparenterally, in formulations containing conventionally acceptablecarriers, adjuvants, and vehicles as desired. The term “parenteral” asused herein includes subcutaneous, intravenous, intramuscular, orintrasternal injection and infusion techniques. Administration includesinjection or infusion, including intra-arterial, intracardiac,intracerebroventricular, intradermal, intraduodenal, intramedullary,intramuscular, intraosseous, intraperitoneal, intrathecal,intravascular, intravenous, intravitreal, epidural and subcutaneous),inhalational, transdermal, transmucosal, sublingual, buccal and topical(including epicutaneous, dermal, enema, eye drops, ear drops,intranasal, vaginal) administration. In some exemplary embodiments, aroute of administration is via an injection such as an intramuscular,intravenous, subcutaneous, or intraperitoneal injection.

Liquid formulations include an oral formulation, an intravenousformulation, an intranasal formulation, an ocular formulation, an oticformulation, an aerosol, and the like. In certain embodiments, acombination of various formulations are administered. In certainembodiments a composition is formulated for an extended release profile.

Methods of Treatment and Use

Disclosed herein, in certain embodiments, are methods of use of Tri-TACsdisclosed herein in the treatment of cancer in an individual in needthereof. In some embodiments, a target-specific ligand of the TACsdisclosed herein bind to a tumor antigen or tumor associated antigen ona tumor cell. In some embodiments, a target-specific ligand of the TACsdisclosed herein selectively bind to a tumor antigen or tumor associatedantigen on a tumor cell. In some embodiments, a target-specific ligandof the TACs disclosed herein specifically bind to a tumor antigen ortumor associated antigen on a tumor cell. In some embodiments, thetarget antigen is a tumor antigen. Examples of tumor antigens include,but are not limited to, CD19, HER-2 (erbB-2), B-cell maturation antigen(BCMA), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125,MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associatedantigen (MAGE), prostate-specific antigen (PSA), glioma-associatedantigen, β-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinalcarboxyl esterase, mut hsp70-2, M-CSF, prostase, PAP, NY-ESO-1, LAGE-1a,p53, prostein, PSMA, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin growthfactor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

Disclosed herein, in certain embodiments, are methods of treating acancer expressing a target antigen in an individual in need thereof,comprising administering to the individual engineered T cells disclosedherein. In some embodiments, the target antigen is CD19. In someembodiments, the method of treating a cancer expressing CD19 in anindividual in need thereof comprises administering to the individualengineered T cells comprising a TAC comprising a CD19-targeting ligand.In some embodiments, examples of cancers that are treated by a TACcomprising a CD19-targeting ligand include, but are not limited to Bcell malignancies. In some embodiments, examples of cancers that aretreated by a TAC comprising a CD19-targeting ligand include, but are notlimited to B cell lymphomas, acute lymphoblastic leukemia (ALL), andchronic lymphocytic leukemia (CLL). In some embodiments, examples ofcancers that are treated by a TAC comprising a CD19-targeting ligandinclude, but are not limited to Non-Hodgkin's lymphoma (NHL).

In some embodiments, the target antigen is HER-2. In some embodiments,the method of treating a cancer wherein a cancer cell expresses HER-2 inan individual in need thereof comprises administering to the individualengineered T cells comprising a TAC comprising a HER-2-targeting ligand.In some embodiments, examples of cancers that are treated by a TACcomprising a HER-2-targeting ligand include, but are not limited tobreast cancer, bladder cancer, pancreatic cancer, ovarian cancer, andstomach cancer.

In some embodiments, the target antigen is BCMA. In some embodiments,the method of treating a cancer wherein a cancer cell expresses BCMA inan individual in need thereof comprises administering to the individualengineered T cells comprising a TAC comprising a BCMA-targeting ligand.In some embodiments, examples of cancers that are treated by a TACcomprising a BCMA-targeting ligand include, but are not limited toleukemia, lymphomas, and multiple myeloma.

Further disclosed herein is use of an engineered T cell disclosed hereinin the preparation of a medicament to treat cancer in an individual inneed thereof. Also disclosed herein is the use of a mixture of T cellscomprising modified and unmodified cells, or comprising differentpopulations of modified cells with or without unmodified cells. One ofordinary skill in the art would understand that a therapeutic quantityof modified T cells need not be homogenous in nature.

In some embodiment, the engineered T cells disclosed herein are part ofa combination therapy. In some embodiments, effectiveness of a therapydisclosure herein is assessed multiple times. In some embodiments,patients are stratified based on a response to a treatment disclosedherein. In some embodiments, an effectiveness of treatment determinesentrance into a trial.

In some embodiments, cancers that are treated engineered T cellscomprising any one of the TAC disclosed herein include any form ofneoplastic disease. In some embodiments, examples of cancers that aretreated include, but are not limited to breast cancer, lung cancer andleukemia, for example mixed lineage leukemia (MLL), chronic lymphocyticleukemia (CLL) acute lymphoblastic leukemia (ALL). In some embodiments,examples of cancers that are treated include, but are not limited tolarge B-cell lymphoma, diffuse large B-cell lymphoma, primarymediastinal B cell lymphoma, high grade B-cell lymphoma, or large B celllymphoma arising from follicular lymphoma. Other cancers includecarcinomas, blastomas, melanomas, sarcomas, hematological cancers,lymphoid malignancies, benign and malignant tumors, and malignancies. Insome embodiments, the cancer comprises non-solid tumors or solid tumors.In some embodiments, cancers that are treated include tumors that arenot vascularized, or not yet substantially vascularized, as well asvascularized tumors. In some embodiments, the cancer is a solid canceror comprises a solid tumor. In some embodiments, the cancer is a liquidcancer or comprises a liquid tumor. In some embodiments, the cancer is alung cancer, a breast cancer, a colon cancer, multiple myeloma,glioblastoma, gastric cancer, ovarian cancer, stomach cancer, colorectalcancer, urothelial cancer, endometrial cancer, or a melanoma. In someembodiments, the cancer is a lung cancer. In some embodiments, thecancer is a breast cancer. In some embodiments, the cancer is a coloncancer. In some embodiments, the cancer is multiple myeloma. In someembodiments, the cancer is a glioblastoma. In some embodiments, thecancer is a gastric cancer. In some embodiments, the cancer is anovarian cancer. In some embodiments, the cancer is a stomach cancer. Insome embodiments, the cancer is a colorectal cancer. In someembodiments, the cancer is urothelial cancer. In some embodiments, thecancer is an endometrial cancer. In some embodiments, the cancer is amelanoma.

TABLE 1 Table of Sequences Nucleotide/ SEQ ID NO Description Amino AcidSEQ ID NO: 1 Tri TAC Configuration 1 Nucleotide SEQ ID NO: 2 Tri TACConfiguration 1 Amino Acid SEQ ID NO: 3 Tri TAC Configuration 2Nucleotide SEQ ID NO: 4 Tri TAC Configuration 2 Amino Acid SEQ ID NO: 5muIgG leader (secretion signal) Nucleotide SEQ ID NO: 6 muIgG leader(secretion signal) Amino Acid SEQ ID NO: 7 DARPin specific for Her2antigen Nucleotide SEQ ID NO: 8 DARPin specific for Her2 antigen AminoAcid SEQ ID NO: 9 Myc Tag Nucleotide SEQ ID NO: 10 Myc Tag Amino AcidSEQ ID NO: 11 Linker 1 Nucleotide SEQ ID NO: 12 Linker 1 Amino Acid SEQID NO: 13 UCHT1¹ Nucleotide SEQ ID NO: 14 UCHT1² Amino Acid SEQ ID NO:15 Linker 2 Nucleotide SEQ ID NO: 16 Linker 2 Amino Acid SEQ ID NO: 17CD4 Domain³ Nucleotide SEQ ID NO: 18 CD4 Domain⁴ Amino Acid SEQ ID NO:19 CD4 based linker Nucleotide SEQ ID NO: 20 CD4 based linker Amino AcidSEQ ID NO: 21 OKT3 Nucleotide SEQ ID NO: 22 OKT3 Amino Acid SEQ ID NO:23 F6A Nucleotide SEQ ID NO: 24 F6A Amino Acid SEQ ID NO: 25 L2KNucleotide SEQ ID NO: 26 L2K Amino Acid SEQ ID NO: 27 Short Helixconnector Nucleotide SEQ ID NO: 28 Short Helix connector Amino Acid SEQID NO: 29 Long Helix connector Nucleotide SEQ ID NO: 30 Long Helixconnector Amino Acid SEQ ID NO: 31 Large domain connector Nucleotide SEQID NO: 32 Large domain connector Amino Acid SEQ ID NO: 33 ScFv specificfor BCMA antigen Nucleotide SEQ ID NO: 34 ScFv specific for BCMA antigenAmino Acid SEQ ID NO: 35 ScFv specific for CD19 antigen Nucleotide SEQID NO: 36 ScFv specific for CD19 antigen Amino Acid SEQ ID NO: 37 CD8αDomain Nucleotide SEQ ID NO: 38 CD8α Domain Amino Acid SEQ ID NO: 39CD8α + R(β) Domain Nucleotide SEQ ID NO: 40 CD8α + R(β) Domain AminoAcid SEQ ID NO: 41 CD8 α + Lck Domain Nucleotide SEQ ID NO: 42 CD8 α +Lck Domain Amino Acid SEQ ID NO: 43 huUCHT1 Nucleotide SEQ ID NO: 44huUCHT1 Amino Acid SEQ ID NO: 45 huUCHT1 (Y177T) Nucleotide SEQ ID NO:46 huUCHT1 (Y177T) Amino Acid SEQ ID NO: 47 huIgG Nucleotide SEQ ID NO:48 huIgG Amino Acid SEQ ID NO: 49 huCD8a Nucleotide SEQ ID NO: 50 huCD8aAmino Acid SEQ ID NO: 51 3625 scFv BCMA Vh-Vl Nucleotide SEQ ID NO: 523625 scFv BCMA Vh-Vl Amino Acid SEQ ID NO: 53 3625 scFv BCMA Vl-VhNucleotide SEQ ID NO: 54 3625 scFv BCMA Vl-Vh Amino Acid SEQ ID NO: 553625 TAC Helix Vh-Vl huUCHT1 Nucleotide SEQ ID NO: 56 3625 TAC HelixVh-Vl huUCHT1 Amino Acid SEQ ID NO: 57 3625 TAC Helix Vl-Vh huUCHT1Nucleotide SEQ ID NO: 58 3625 TAC Helix Vl-Vh huUCHT1 Amino Acid SEQ IDNO: 59 3625 TAC G4S Vh-Vl huUCHT1 Nucleotide SEQ ID NO: 60 3625 TAC G4SVh-Vl huUCHT1 Amino Acid SEQ ID NO: 61 3625 TAC G4S VL-VH huUCHT1Nucleotide SEQ ID NO: 62 3625 TAC G4S VL-VH huUCHT1 Amino Acid SEQ IDNO: 63 CD19-TAC Nucleotide SEQ ID NO: 64 CD19-TAC Amino Acid SEQ ID NO:65 huIgG Her2 TAC huUCHT1 Nucleotide SEQ ID NO: 66 huIgG Her2 TAChuUCHT1 Amino Acid SEQ ID NO: 67 CD8a Her2 TAC huUCHT1 Nucleotide SEQ IDNO: 68 CD8a Her2 TAC huUCHT1 Amino Acid SEQ ID NO: 69 Flexible ConnectorAmino Acid SEQ ID NO: 70 Flexible Connector Nucleotide SEQ ID NO: 71UCHT1 (Y182T) Nucleotide SEQ ID NO: 72 UCHT1 (Y182T) Amino Acid ¹Lightchain, nucleotides 1-324; Linker, nucleotides 325-387; Heavy chain,nucleotides 388-750 ²Light chain, amino acids 1-108; Linker, amino acids109-128; Heavy chain, amino acids 129-250 ³Extracellular linker,nucleotides 1-66; Transmembrane domain, nucleotides 67-132; Cytosolicdomain, nucleotides 133-254 ⁴Extracellular linker, amino acids 1-22;Transmembrane domain, amino acids 23-44; Cytosolic domain, amino acids45-84

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1. Characterization of the Tri-TAC Technology

An overview of the Tri-TAC technology is provided in FIG. 1A-FIG. 1C.

FIG. 1A shows an example of CD8 T-cell activation based on theco-assembly of different receptors and their associated proteinpartners. Initially, the major histocompatibility complex I ispresenting an antigen (helix). This is recognized by a T cell receptor(TCR) complex capable of binding the antigen. The TCR complex containsseveral individual subunits. The α/β domains are able to interactdirectly with the antigen presented on MHC-I. The α/β domains theninteract with several other domains (ε, γ, δ, and ζ), all of whichparticipate in T-cell activation via various intracellular activationdomains. The TCR complex interacts with MHC-I concurrently with the CD8co-receptor. The CD8 co-receptor binds to the MHC-I in an antigenindependent manner. CD8 directly interacts with Lck, a protein kinaseimportant for activating the TCR receptor complex. The CD8 and Lckinteraction also ensures their association with lipid rafts (membraneportion) microdomains, which are hypothesized to organize andencapsulate other relevant signaling moieties (dark spheres). Laterstages of activation then lead to CD28 recruitment. If this interactioncascade occurs several times in parallel, T-cells become activated andare able to exert their cytotoxic effects.

FIG. 1B provides an overview of Chimeric Antigen Receptors (CAR). CARsseek to reproduce the complex mechanism of T-cell activation bycombining several key activation domains, such as CD3ζ and CD28 in asingle synthetically engineered molecule. The CAR then directlyinteracts with an antigen of choice using specific binding domains.Depicted here is an ankyrin repeat protein (DARPin). It is believed thatseveral such interactions occurring in parallel lead to T-cellactivation.

FIG. 1C is an overview of the Tri-TAC technology mimicking the naturalactivation process. The Tri-TAC was developed to better recapitulate thenatural signaling through the TCR, while retaining MHC unrestrictedtargeting. T-cell activation occurs following ligation of MHC by the TCRand T cell co-receptor (either CD4 or CD8), which simultaneously bind toconserved regions within the MHC molecule. The co-receptors arespecifically located within “lipid rafts”, membrane micro domains thatare particularly important for TCR signal complex formation. In additionto ensuring the correct microdomain localization of the TCR activationcomplex, these co-receptors also bind directly to Lck, a protein kinasethat is crucial for T-cell activation. None of the traditional chimericreceptors or bi-functional proteins engage the co-receptor molecules orLck. A molecule was created where the transmembrane and intracellularregions of the CD4 co-receptor, which localize to the lipid raft andbind Lck, respectively, were fused to single-chain antibody that bindsCD3 (UCHT1; SEQ ID NO: 13, 14 and homologs thereof). This construct isdesigned to draw the CD3 molecule and the TCR into regions of lipidrafts and bring Lck into the proximity of the TCR, similar to naturalMHC binding. To target this receptor, a designed ankyrin repeat (DARPin)was linked to the CD4-UCHT1 chimera to generate a Trifunctional Tcell-antigen coupler (Tri-TAC). In this example, the DARPin was specificfor the proto-oncogene, HER-2 (erbB-2).

Multiple Tri-TAC configurations are possible (FIG. 2A and FIG. 2B). Inconfiguration 1 (FIG. 2A) the Antigen binding domain is locatedN-terminal, connected to the CD3 ligand binding domain and then theco-receptor domain. In configuration 2 (FIG. 2B) the CD3 ligand bindingdomain is located N-terminal, connected to the antigen binding domainwhich in turn connects to the co-receptor domain.

Multiple classes of ligand binding domains can be incorporated into theTri-TAC molecule (FIG. 3A-FIG. 3D). The examples herein illustrate ageneral schematic of a configuration 1 Tri-TAC (FIG. 3A), a Tri-TACbearing a HER-2-specific DARPin (FIG. 3B), a Tri-TAC bearing aCD19-specific scFv (FIG. 3C), and a Tri-TAC bearing a BCMA-specific scFv(FIG. 3D).

FIG. 4A-FIG. 4D illustrate the functionality of a Tri-TAC bearing theHER-2-specific DARPin. Human T cells were engineered to express eitherthe Tri-TAC as disclosed herein or a conventional CAR with the sameDARPin. It was determined that in all aspects, T cells engineered withthe Tri-TAC demonstrated functionality at least equivalent to aconventional CAR. Interestingly, with regard to 2 parameters (TNF-αproduction and CD107a mobilization), it was observed that the Tri-TACwas more active than a conventional CAR in some circumstances.

FIG. 4A shows surface expression of Anti-HER-2 DARPin Tri-TAC comparedto Anti-HER-2 DARPin CAR, and control T cells. The chimeric receptorswere detected by incubation with recombinant HER-2. The Anti-HER-2DARPin Tri-TAC was expressed well on the surface of the engineered Tcells. FIG. 4B shows growth of the engineered T cells cultures. T cellswere activated with anti-CD³/anti-CD28 Dynabeads and engineered withlentiviruses encoding the Tri-TAC, CAR or no receptor (control). After 2weeks, the CAR and control cultures had grown to similar numbers whilethe Tri-TAC cultures grew slightly more slowly. FIG. 4C and FIG. 4D showthe functional attributes of the engineered T cells. T cells engineeredto express the Tri-TAC or the CAR bearing the HER-2 DARPin werestimulated with plate-bound antigen. The T cells engineered to expressthe Tri-TAC and CAR could elaborate all measured functions (TNF-αproduction, IFN-γ production and CD107a mobilization, FIG. 3C and FIG.3D). T cells engineered with the Tri-TAC exhibited elevated frequenciesof CD107a-positive cells following stimulation relative to T cellsengineered with a CAR (FIG. 3D), suggesting enhanced cytotoxicity on aper-cell basis.

FIG. 6A-FIG. 6J provides data confirming the importance of both ligandbinding domain and the UCHT1 CD3 binding domain for Tri-TACfunctionality. T cells were engineered with the full-length Tri-TACbearing the HER-2 DARPin (FIG. 6G, FIG. 6H, FIG. 6I, bottom row), aTri-TAC variant that lacks the DARPin (FIG. 6A, FIG. 6B, FIG. 6C, toprow), or a Tri-TAC variant that lacks the UCHT1 (FIG. 6D, FIG. 6E, FIG.6F, middle row). All three engineered T cell populations were stimulatedwith HER-2-positive tumor cells. The T cells engineered with thefull-length Tri-TAC could produce IFN-g, TNF- and IL-2 followingstimulation, whereas the variants failed to produce any cytokinefollowing stimulation. The three T cell populations were alsoco-cultured with D2F2/E2 cells (HER-2-expressing) or D2F2 cells(HER-2-negative) at an effector: target of 4:1 (FIG. 6J). T cellsengineered with full-length Tri-TAC demonstrated robust killing againstD2F2/E2 cells but did not kill the D2F2 cells. The other Tri-TACvariants lacking either the DARPin or the UCHT1, exhibited no killing.

FIG. 7A-FIG. 7C show the results of mice treated with vector control(NGFR), Anti-HER-2 DARPin CAR or Anti-HER-2 DARPin Tri-TAC. A xenograftmouse model was used. OVCAR-3 tumor cells were administered to micesubcutaneously and allowed to grow until the tumors reached a size of100-200 mm³. FIG. 7A shows relative tumor progression normalized totumor size at day of treatment. Anti-HER-2 DARPin Tri-TAC engineeredT-cells caused a rapid decrease in tumor volume, control had no effect,and CAR cells slowed tumor growth and showed a delayed reduction intumor size. FIG. 7B illustrates relative changes in body weight postT-cell infusion. Both control and anti-HER-2 DARPin Tri-TAC engineeredcells show no significant changes in mouse body weight post treatment.In contrast, Anti-HER-2 DARPin CAR-treated mice show significant loss inbody weight indicative of severe toxicity. FIG. 7C illustrates cytokineconcentrations in serum of mice on day 7 post T-cell infusion. Cytokinelevels were higher in CAR-treated mice compared to Tri-TAC-treated mice.

Example 2. Substitutions of UCHT1 Influence Tri-TAC Function

FIG. 8A-FIG. 8H illustrate the functionality of Tri-TACs bearingalternate CD3 binding domains. The domains are listed in FIG. 8A andFIG. 8E. Tri-TACs containing UCHT1 (FIG. 8B), OKT3 (FIG. 8B) and huUCHT1(FIG. 8F) displayed high surface expression, whereas the Tri-TACscontaining F6A (FIG. 8F) and L2K (FIG. 8F) revealed lower surfaceexpression. Cells expressing the Tri-TAC containing OKT3 exhibited lowcytokine production (FIG. 8C, FIG. 8C1) and intermediate cytotoxicity(FIG. 8D) upon Tri-TAC ligation. Cells expressing the Tri-TAC containingF6A exhibited strong cytokine production (FIG. 8G, FIG. 8G1) andcytotoxicity (FIG. 8H) following Tri-TAC ligation. Cells expressing theTri-TAC containing L2K exhibited low cytokine production (FIG. 8G, FIG.8G1) and intermediate cytotoxicity (FIG. 8H).

FIG. 9A-FIG. 9H illustrates TCR surface expression on T cells engineeredwith different Tri-TAC variants shown in FIG. 8A and FIG. 8E. T cellsengineered with the Tri-TAC variants comprising OKT3 (FIG. 9A, FIG. 9Eand FIG. 9B, FIG. 9F) or L2K (FIG. 9C, FIG. 9G and FIG. 9D, FIG. 9H)exhibited lower TCR surface expression relative to the T cellsengineered with Tri-TACs comprising UCHT1 or huUCHT1, respectively. Incontrast, T cells engineered with the Tri-TAC variant comprising F6A didnot reveal TCR downregulation relative to the Tri-TAC carrying huUCHT1(FIG. 9C, FIG. 9G and FIG. 9D, FIG. 9H). The F6A substitution reducedTri-TAC receptor surface expression, while retaining moderate cytokineproduction and cytotoxicity. The L2K substitution moderately reducedsurface expression and reduced cytokine production, but retainedintermediate cytotoxicity. The OKT3 substitution resulted in highTri-TAC surface expression, low cytokine production, and intermediatecytotoxicity. These data indicate that Tri-TAC surface expression and Tcell effector functions are not inherently proportional, and thatTri-TAC domain substitutions, in some instances, alters effectorfunctions independent of surface expression levels. It is conceivablethat a TAC variant with reduced cytotoxicity and low surface expressioncould be of value in certain clinical applications.

In many cases, the scFv substitutions attenuated the ability of theengineered T cell to elaborate IFN-γ, TNF-α, and IL-2, yet theengineered T cells retained the ability to kill target cells. Excessivecytokine production has been associated with adverse events in clinicalsettings, limiting current CAR technologies to life-threateningdiseases. The ability to modify TAC molecules to reduce their cytokineproduction while retaining moderate cytotoxicity will allow generationTri-TAC receptors with the exact level of reactivity required to satisfyclinical efficacy and safety.

The capacity of the Tri-TAC variant comprising OKT3 to suppress TCRsurface expression and cytokine production, while retainingcytotoxicity, could be of great value in allogeneic situations where thesuppression of TCR could suppress graft versus host disease.

These data demonstrate that scFv substitutions of UCHT1 influences thefunction of Tri-TACs. Further modifications will be result in Tri-TACsuseful in various applications (e.g., oncology, autoimmunity, allergy).

Example 3. Introducing Various Linkers Connecting the Ligand that Bindsa TCR Complex to the Target-Binding Ligand Domain

FIG. 10A-FIG. 10B illustrate several TAC variants with different linkersconnecting the ligand that binds a TCR complex and the target-bindingligand domain. The flexible connector allows movement between the twodomains. The large domain connector contains two folded domains and isvery large and rigid. The small and long helix connectors also introducerigidity but are less restrictive when compared to the large domainlinker.

FIG. 11A-FIG. 11E illustrate the impact of connector substitution onTri-TAC surface expression, Tri-TAC transduction efficiency, andcytokine production upon Tri-TAC ligation. FIG. 11A and FIG. 11B showthat the helical linkers enhance surface expression and transductionefficiency when compared to the flexible linker, while the large domainconnector enhances transduction efficiency but not surface expression.FIG. 11D, FIG. 11E illustrates cytokine production by cells expressingTri-TACs with short helix, long helix, or large domain connectors.

FIG. 12A illustrates enhanced in vitro cytotoxicity of T cellsexpressing Tri-TACs with the short helix connector. FIG. 12B illustratesenhanced in vivo tumor control of T cells expressing Tri-TACs with theshort helix connector. The short helical connector was associated withhigh in vitro cytotoxicity and effective in vivo tumor control.

Example 4. Introducing a CD8α/β Cytosolic Domain

FIG. 13A illustrates surface expression of CD8α Tri-TAC paired with ananti-HER-2 scFv or FIG. 13C anti-HER-2 DARPin. FIG. 13B illustratescytokine production by T cells expressing CD8α Tri-TAC paired with ananti-HER-2 scFv or anti-HER-2 DARPin.

FIG. 14A illustrates a CD4 Tri-TAC monomer and a CD8α/β heterodimer. TCRco-receptors, both CD4 and CD8, carry functional domains that areimportant for the co-receptor functionality. These regions include thearginine rich region that is hypothesized to be important for lipid raftassociation, and the CXCP motif required for Lck binding. Unlike CD4,which is a monomer, the CD8 co-receptor is a heterodimer composed of anα and a β subunit (FIG. 14A). Both the α and β CD8 subunits containarginine rich regions, but only the a subunit contains the CXCP motif.

FIG. 14B-FIG. 14D provide schematics of Tri-TAC variants thatincorporate elements from the CD8 co-receptor shown in FIG. 14A. Thecysteine responsible for dimerizing CD8α and CD8β was replaced with analanine in all CD8 Tri-TAC variants. FIG. 14B is a schematic of a CD8αTri-TAC comprising a Cysteine to Serine mutation to ensure a monomericreceptor distribution, and a CD8α cytosolic domain. FIG. 14C is aschematic of a CD8α+Rβ Tri-TAC comprising a Cysteine to Serine mutationto ensure a monomeric receptor distribution, and a chimeric CD8αcytosolic domain where the CD8α arginine rich region is replaced withthe CD8β arginine rich region. FIG. 14D is a schematic of a CD8β+LckTri-TAC comprising a Cysteine to Serine mutation to ensure a monomericreceptor distribution, and a chimeric CD8β cytosolic domain, where theCD8α CXCP domain, which contains an Lck binding motif, was added to theC-terminus of the CD8β cytosolic domain.

FIG. 15A-FIG. 15D illustrate various phenotypic and functionalattributes of the CD8-based Tri-TAC variants relative to theprototypical Tri-TAC. FIG. 15A-FIG. 15B illustrate surface expression ofCD8-Tri TAC variants relative to the prototypic Tri-TAC. Surfaceexpression was comparable among the different Tri-TACs. FIG. 15Cillustrates in vitro cytotoxicity of CD8-Tri TAC variants co-culturedwith LOX IMVI (HER-2 negative) and A549, SKOV3, SKBR3 or MBA MB 231 (allare HER-2 positive). All T cells engineered with Tri-TACs exhibitedcytotoxicity. FIG. 15D illustrates cell division of T cells engineeredwith either the CD8 Tri-TAC variants or the prototypic Tri-TAC (FIG.15D). FIG. 15E illustrates TCR surface expression of engineered T cellscomprising CD8 Tri-TAC variants or the prototypic Tri-TAC. All Tri-TACvariants had a similar effect on TCR expression. While the CD4co-receptor demonstrated good surface expression and functionality withboth the scFv and DARPin anti HER-2, the CD8α construct showed activityonly in the context of the DARPin antigen binding domain. When testingdifferent CD8α cytosolic domains, all the configurations contained thereported key sequence attributes associated with co receptorfunctionality (Arginine rich region and CXCP). All CD8α/β constructsshowed similar performance when compared to the CD4 prototype. Thisemphasizes that the retention of specific biochemical properties, suchas lipid raft affinity and Lck binding, is more important to determineTri TAC performance than a specific cytosolic polypeptide sequence.

The growth of T cells engineered with the CD8α+R(β) and the CD8β+LckTri-TACs was significantly impaired relative to the growth of T cellsengineered with the other variations. Despite a significant impact ongrowth, these Tri-TACs all displayed a comparable ability to activate Tcells. The reduced growth of the CD8α+R(β) and the CD8β+Lck Tri-TACs maybe advantageous for certain application where maximal T cell expansionis not desirable.

Example 5. Development of a CD19-TAC Construct

FIG. 16 illustrates the step-wise development of a CD19-TAC construct.Several generations of lentiviral vectors are created with variousalterations in design elements to ensure CD19-specificity, proper TACexpression, and GMP-grade lentivirus production. Each box represents alentiviral vector and specifies the 3 major design elements: (A) theantigen-binding domain, (B) the TCR/CD3-binding domain, and (C) theco-receptor domain. Shaded areas indicate domains that have been thesubject of modification during the vector development process.

The TAC in the first step comprises a HER-2-specific designed ankyrinrepeat protein (DARPin), a murine UCHT1 CD3-specific scFv, and aflexible transmembrane and cytosolic CD4 polypeptide. The TAC is clonedinto a pCCL4 lentiviral vector.

To generate a CD19-specific Tri-TAC, the HER-2-specific DARPin wasreplaced with a polypeptide comprising an N-terminal CD8α leader peptidefused to an anti-CD19 scFv. The heavy and light chains of the CD19 scFvwere connected via glycine-serine linker region.

The UCHT1 domain was replaced with a humanized version (huUCHT1) toreduce immunogenicity. This TAC construct exhibited superior surfaceexpression levels than its precursor.

To further improve receptor expression on the cellular surface of Tcells without impairing functionality, two separate modifications wereevaluated in parallel. To increase single chain stabilization, the G₄Slinker (SEQ ID NO: 73) used in the anti-CD19 scFv was replaced with themore structured Whitlow linker. Separately, a Y177T mutation wasintroduced into the huUCHT1 domain. Both strategies enhanced theexpression of the TAC receptor, and a receptor was generated with boththe Whitlow linker and the Y177T mutation.

FIG. 17 illustrates a CD19-TAC insert in a pCCL lentiviral vector. ThepCCL vector features a bi-directional promoter system with ΔNGFR(hu)under control of the mCMV promoter and TAC expression being driven bythe EF-1α promoter. The ΔNGFR(hu) is a truncated human CD271 (Tumornecrosis factor receptor superfamily member 16), with transmembranedomain but lacking the cytosolic signaling domain. The ΔNGFR(hu)expression product is used to quantify lentiviral transduction. TheCD19-TAC #921 open reading frame is enlarged to show the key elements ofthe TAC construct: The CD8α leader, FMC63 single chain (anti-CD19 scFv),the human c-Myc Tag, the huUCHT1 (Y177T) and the ΔCD4 domain. ThehuUCHT1 (Y177T) mutation was identified by examining point mutationsrandomly introduced into resides of the murine UCHT1 CD3 epsilon bindinginterface. In a screen the (Y177T) mutation was successfully identified.The (Y177T) mutation results in better Tri TAC surface expression whileretaining T cell activation. ΔCD4 lacks the four CD4 extracellularimmunoglobulin like domains and retains the extracellular linker,transmembrane and cytosolic domains.

To generate a GMP-grade lentiviral vector, the CD19-Tri-TAC constructwas cloned into a new lentiviral vector under the control of a MSCVpromoter. The CD19-Tri-TAC construct is the same as shown in FIG. 17.

Example 6. Ability to Manufacture CD19-TAC-Expressing T Cells fromDifferent Donor Material

FIG. 18 illustrates the efficacy of CD19 TAC-expressing T cellsmanufactured from multiple donors. CD19-TAC-expressing T cells wereproduced using T cells from three different donors, and tested in theNALM-6 tumor model. Mice bearing established NALM-6 tumors were treatedwith a single dose of 4×10⁶ CD19 TAC-expressing T cells. Control miceshowed rapid tumor outgrowth, with all mice reaching endpoint by thetermination of the study. T cell products from Donors 1 & 2 resulted incomplete control in all mice. T cell product from Donor 3 resulted inrobust tumor control in all mice and long-term control in 2/4 treatedmice. The study confirms that tumor rejection is achieved by CD19TAC-expressing T cells derived from multiple healthy donors. The resultsof the NALM-6 tumor model in FIG. 18 suggest that efficacious CD19 TACis produced from multiple donor source materials.

Example 7. In Vitro Cytotoxicity and In Vivo Efficacy ofCD19-TAC-Expressing T Cells

To evaluate the ability of CD19-TAC to effectively engage variousCD19-positive cells, Tri-TAC-engineered T cells were co-cultured witheither NALM-6 (acute lymphoblastic leukemia), Raji (Burkitt lymphoma) orJeko-1 (Mantle Cell Lymphoma). NALM-6, Jeko-1 and Raji cells wereengineered with enhanced firefly luciferase to enable tracking of tumorburden in vitro and in the live animal via bioluminescence imaging.

FIG. 19A-FIG. 19C illustrates killing of tumor cell lines byCD19-TAC-expressing T cells. The effects were dose-dependent andincreased with increasing effector-to-target (E:T) ratios. As negativecontrols, cells engineered with ΔTAC (lacking an antigen-binding domain)or non-transduced T cells were used. These results demonstrate thatCD19-TAC-expressing T cells kill CD19-positive tumor cells.

FIG. 19 D-FIG. 19G illustrates the design and outcome of an in vivostudy assessing efficacy of CD19-TAC in mice engrafted with eitherNALM-6 (acute lymphoblastic leukemia), Raji (Burkitt lymphoma) or Jeko-1(Mantle Cell Lymphoma) liquid tumors. To initiate NALM-6, Raji andJeko-1 tumors, mice were inoculated with NALM-6, Raji or Jeko-1 cellsand housed 4 or 7 days, respectively, to allow the engraftment oftumors. On day 4 or 7, CD19-TAC-expressing T cells were given as anintravenous tail vein injection. Tumor burden was measured at weeklyintervals, and the data are plotted as the average radiance[p/s/cm{circumflex over ( )}2/sr].

FIG. 19E-FIG. 19G illustrates that CD19-TAC engineered T cells areefficacious in inducing tumor regression and long-term tumor control inNALM-6 (acute lymphoblastic leukemia), Raji (Burkitt lymphoma) or Jeko-1(Mantle Cell Lymphoma) liquid tumors.

The results of the NAML-6, Raji or Jeko-1 tumor models in FIG. 19A-FIG.19G suggest that CD19-TAC is efficacious in a variety of CD19 positivetumor models.

Example 8. CD19-TAC-Expressing T Cell Persistence and Lasting TumorImmunity

FIG. 20A-FIG. 20B illustrate persistence of tumor immunity andresistance to re-challenge in mice receiving CD19-TAC-expressing Tcells. Mice bearing established NALM-6 tumors were treated withCD19-TAC-expressing T cells.

FIG. 20A illustrates the experimental set up to determine CD19-TACpersistence in mice. Mice were first inoculated with NALM-6 cells, whichfollowing a 4 day engraftment period were treated with CD19-TAC. Allmice showed tumor regression and complete tumor control. 56 days afterthe initial treatment mice were re-challenged with either NALM-6 (CD19positive) or KMS11 (CD19 negative) liquid tumors. In all cases naïvemice are co-injected with tumor cells and used as negative controls.Tumor burden is followed via luminescence signal.

FIG. 20B: Mice bearing established NALM-6 tumors were treated withCD19-TAC-expressing T cells given as split dose totaling 4×10⁶engineered cells. As controls, a group of un-treated animals was used.Following ACT, treated mice presented durable anti-tumor responses. Incontrast, control mice showed exponential increases in tumor masses andreached tumor burden related endpoint. On day 56 post-ACT, mice werere-challenged with either NALM-6 tumor cells (CD19 positive) or KMS11tumor cells (CD19 negative). CD19-TAC-treated mice remain protected fromNALM-6 (CD19 positive) tumor cells, but not from KMS11 (CD19 negative)tumor cells.

The results of re-challenge experiments in FIG. 20A and FIG. 20B suggestthat CD19-TAC, in some instances, differentiates into long-lived memorycells that retain anti-tumor properties.

Example 9. In Vivo Expansion and Dose Dependency of CD19-TAC-ExpressingT Cells

FIG. 21 and FIG. 22 illustrate dose dependency, dose regime (split orsingle) and expansion of CD19-TAC-expressing T cells in a NALM-6 cancermodel. FIG. 21A illustrates experimental design. Mice received either asingle dose of CD19-TAC-expressing T cells on day four post-tumorinoculation, or a split dose delivered seven days apart. MultipleCD19-TAC-expressing T cells doses were tested: 0.5×10⁶, 1×10⁶, and 4×10⁶cells. FIG. 21B control groups of mice receive 4×10⁶ non-transducedcells, or freezing media (vehicle control).

FIG. 21B illustrate survival of mice after NALM-6 injection and CD19-TACinjection. Dose-dependent promotion of survival were observed both inthe single dose and split dose groups, with the highest singleadministration dose limiting tumor growth and promoting survival themouse.

FIG. 22A illustrates the gating strategy used to assess T cellproliferation. Cells were first selected based on forward and sidewaysscatter to select for the lymphocyte population. Singlet cells wereidentified via a forward scatter area over height gate. Live cells wereidentified via near IR gating. Human cells were identified via a hCD45gate. The resulting subset of cells was further divided into CD3positive cells. These cells were then gated on CD4/CD8 and Protein L.The staining strategy also contained muCD45-1 to identify murine bloodcells. CD19 was included to stain for NALM-6 cells.

FIG. 22B expansion of T cells in mice after split dose adoptive T celltransfer (ACT). After ACT, blood samples were taken regularly andanalyzed via flow cytometry. Values were normalized to the number oftotal T cells present in the blood post ACT1. Values were alsonormalized to the total number of CD45.1+(murine) cells to account fordifferences in blood draw. T cells in mice treated with CD19-TACengineered cells were shown to expand in recipient mice withinapproximately 1-2 weeks after the first ACT (FIG. 22B). Non-transducedcells did not expand (FIG. 22B).

The results of the various doses, dose regimen (FIG. 21B) and T cellcounts (FIG. 22B) suggest that CD19-TAC efficacy is dose dependent, thatengineered T cells expand in vivo, and that this expansion is specificto CD19-TAC engineered cells in animals carrying CD19-positive tumors.

Example 10. In Vivo Efficacy, Long Term Efficacy and Safety CD19-TACTreatment

FIG. 23-FIG. 25 demonstrate the long-term safety and efficacy (FIG. 23)and in the absence of any acute treatment associated toxicities (FIG.24-FIG. 25).

FIG. 23A illustrates the experiment design. Mice were injected with0.5×10⁶ enhanced luciferase engineered NALM-6 cells, which were allowedto engraft for 4 days. Mice are then treated with two dose levels (4 and12×10⁶ engineered cells) of CD19-TAC-engineered T cells in a single doseadministration. Tumor growth was then followed via regular luminescemeasurements. Mouse health was regularly assessed via inspection ofmouse behavior and physical characteristics (grooming, motility, furintegrity)

FIG. 23B illustrates the tumor burden via luminescence followingtreatment with either vehicle alone (Freezing media), non-engineeredcontrol cells (Total T cell dose equal to total T cell dose of highestengineered treatment group) and either 4 or 12×10⁶ engineered CD19-TACengineered T cells. Both controls show rapid tumor outgrowth and noanti-tumor efficacy. The control dose results in a delay in tumoroutgrowth relative to vehicle alone, presumably due to competitionbetween high dose T cell and tumor cells for engraftment niches.Engineered T cell show tumor regression in all cases. High dosetreatment groups show complete tumor control in all cases. The 4×10⁶treatment group shows 3 mice with complete control, one with delayedtumor outgrowth and one with controlled but high tumor burden.

FIG. 23C illustrates overall survival of the different treatment groups.In both, the vehicle and non-engineered control mice, all mice succumbto the tumor within 23 to 35 days respectively. In case of high doseCD19-TAC treatment all mice develop GvHD symptoms and succumb to GvHDwithin 61 days. GvHD is a consequence of the mouse model itself and notthe treatment with the modified T cells. Low dose mice show survival of3 mice until end of study at 90 days, one mouse succumbs to high tumorburden, one mouse succumbs to GvHD.

FIG. 24 and FIG. 25 illustrates clinical chemistry parameters andcytokine levels from vehicle control, non-engineered and CD19-TAC (4 and12×10⁶ effective CD19-TAC engineered cells) treated mice. Mice werefollowed for 33 days with blood samples taken 5, 12 and 33 days postACT. Only CD19-TAC treated mice survived for 33 days. Vehicle controlmice succumbed to tumor burden before a 3^(rd) blood sample could becollected, non-engineered cells were sacrificed early on day 26,immediately prior to mice reaching tumor burden related endpoint. Allblood samples were analyzed for several clinical chemistry parametersand cytokine levels.

FIG. 24 illustrates that at day 5 and 12 CD19-TAC treated mice show noparameter that is significantly higher compared to control groups. Atday 33 all treated mice show clinical chemistry parameters comparable toearly treatment time points, with the exception of AlanineAminotransferase (ALT) and Aspartate Aminotransferase (AST) where somemice experience high levels, similar to mice treated with non-engineeredcells sampled on day 26.

FIG. 25 illustrates the cytokine response on day 5, 12 and 33. On day 5post ACT CD19-TAC but not control mice show elevation in all cytokinestested. The cytokine increase is in agreement with an inflammatoryresponse of CD19-TAC engineered T cells recognizing and reacting toantigen positive NALM-6 tumor cells. Following their initial reaction byday 12 cytokine levels subside, which correlates with the by theninduced tumor regression and generally low tumor burden. On day 12cytokine levels between CD19-TAC treated are either similar or lowerthan non-engineered T cells except for IL10. At the later stage all micetreated with non-transduced or CD19-TAC engineered T cells show anincrease in cytokines, presumably associated with GvHD onset. See alsoFIG. 29, which illustrates cytokine response on day 5, 12, 26 and 33.

The results of the long-term follow up of mice treated with CD19-TAC andtheir clinical chemistry profile demonstrate that engineered T cells aresafe to use and do not show any indication of toxicity causedspecifically by CD19-TAC engineering. The results of the cytokine studydemonstrate an early inflammatory response associated with anti-tumorefficacy, following by a drop in all cytokine levels, suggesting that acontrolled inflammatory response.

Example 11. In Vivo Efficacy of Several BCMA Tri-TAC Variants

FIG. 26 illustrates an in vivo efficacy study of various BCMA Tri-TACconstructs. FIG. 26A illustrates the overall experimental design. 1million luciferase-engineered KMS11 (BCMA positive) tumor cells wereallowed to engraft for 12 days. Mice were then treated with a singleeffective dose of 4 million BCMA constructs and controls (FIG. 26B).Tumor burden was regularly assessed via luminescence measurements. Allmice that showed tumor regression and tumor control were thenre-challenged on day 25 post ACT with 1 million KMS11 cells.

FIG. 26C: Following ACT, control mice exhibited a rapid outgrowth oftumor cells reaching tumor associated endpoint within 19 to 25 days. Incontrast all BCMA-TAC treated mice showed initial tumor regression.Tumor control varied across constructs with the G₄S (SEQ ID NO: 73)3625VH-VL showing the lowest level of initial tumor control and ShortHelix 3625 VL-VH showing the highest level of initial tumor control.Following re-challenge, a majority of all constructs that had maintainedtumor control until day 25 remained protected against re-challenge.

The results of this in vivo study demonstrate, that a variety of BCMATri-TAC constructs are effective in controlling KMS11 (BCMA positive)liquid tumors. But that certain preferred configurations providesuperior efficacy. In general, the helical connector region provided arelative benefit when compared to the flexible linker within the samescFv configuration.

Example 12. In Vivo of TAC-Her2

Mice are inoculated at the hind flank with OVCAR3 solid tumors. Tumorsare allowed to establish and grow to a size of 100 mm³. Mice are thentreated with a tail vain injection of TAC-Her2 engineered T cells. Tumorvolume is measured regularly.

Example 13. Clinical Trial

A clinical study is undertaken wherein subjects of at least 18 years ofage with CD19-positive Diffuse Large B-cell Lymphoma who have failed atleast two prior lines of therapies including ASCT or who are ineligiblefor ASCT are treated with CD19-TAC-expressing T cells. The study is anopen label, single arm, Phase 1/2 two-stage trial, featuring a doseescalation stage to determine the maximum tolerated dose (MTD) orrecommended phase II dose (RPh2D), followed by an expansion cohort atthe selected dose.

Upon enrollment, subjects undergo leukapheresis to obtain T cells formanufacture of CD19-TAC-expressing T cells. Upon successful manufacture,subjects enter the treatment phase. This phase involves alymphodepleting chemotherapy with fludarabine and cyclophosphamide,followed by intravenous (IV) administration of CD19-TAC-expressing Tcells. After treatment with CD19-TAC-expressing T cells, subjects enterpost-treatment follow-up and are followed for safety, disease status,and survival for 2 years after their last dose of CD19-TAC-expressing Tcells. After study completion, subjects are followed for survival,long-term toxicity, and viral vector safety in a separate long-termfollow up protocol for up to 15 years after their last dose ofCD19-TAC-expressing T cells.

In all groups, safety is assessed throughout the study. T cell expansionis assessed from the time of the first dose of CD19-TAC-expressing Tcells until cells are no longer detectable. Radiographic diseaseassessment is performed by positron emission tomography (PET) and/orcomputed tomography (CT) scans pre-treatment and approximately 3, 6, 9,12, 18, and 24 months following the last dose of CD19-TAC-expressing Tcells, or until progressive disease, or treatment with additionalanti-cancer therapy.

Example 14. Manufacturing of CD19-TAC-Expressing T Cell Drug Products

The manufacturing process of CD19-TAC-expressing T cells drug productsinvolves selecting CD4/CD8 T cells from a leukapheresis product,activating the CD4/CD8 positive cells, transducing the cells with alentiviral vector comprising the CD19-TAC construct (as described inexample 5), expanding the transduced cells to level adequate for theproposed dosing schedule, and harvesting and cryopreserving the finalproduct.

The patient's leukapheresis material with its associated unique subjectidentifier (UPN) is received into a manufacturing site and given aunique specimen number (ISN). The CD4/CD8 cells are selected arecryopreserved until initiation of the culture process steps.

The cryopreserved CD4/CD8 positively selected T cells are thawed at 37°C., resuspended in appropriate medium and seeded into culture bags withactivating reagents, the cultures are incubated overnight at 37° C./5%CO2.

The cells are transduced with the CD19-TAC lentiviral vector at anappropriate multiplicity of infection (MOI) and incubated overnight at37° C./5% CO2. On subsequent days, the culture is supplemented withcomplete medium to maintain a desired cell concentration and eventuallypooled into transfer bags, pelleted, resuspended and seeded to largerculture bags at the targeted cell density.

For drug product formulation, the harvested cell suspension isresuspended in excipient and cryopreserved with a controlled-ratefreezer then transferred to LN2 storage.

The product is shipped to the clinical site in its frozen state, thawedat the bedside and administered intravenously.

Prior to the clinical trial, engineering manufacturing runs areconducted inclusive of all in-process and release testing using healthydonor leukapheresis material. In addition to in-process and releasetesting, studies supportive of regulatory filings are conducted on finaldrug product from these engineering runs. These studies includepost-thaw stability, initiation of long-term stability, residual testingto assure the clearance of growth-promoting cytokines and earlyassessment of potential functional/potency indicating assays.

Example 15. Preclinical Development of BCMA Specific T-Cell AntigenCoupler (TAC) Therapy for the Treatment of BCMA Positive Malignancies

FIG. 27 illustrates that TACs proliferate when encountering antigen oncells, but not when the antigen is presented on artificial beads; butCARs proliferate irrespective if antigens are presented on beads orcells.

FIG. 28A-FIG. 28B illustrate TAC engineered T cells expand in vivo andprovide long term protection, indicating cell persistence in a model ofmyeloma. FIG. 28A-FIG. 28B illustrate BCMA-TAC T cells reject multiplemyeloma tumors in a KMS-11 xenograft model engineered with NanoLuc (KMS11-NanoLuc) (BCMAP″). Following tumor engraftment mice were treated withBCMA TAC-T cells (carrying Firefly Luciferase). TAC-T cells expandsignificantly following administration. This correlates with tumorregression. Treated mice were resistant to tumor rechallenge indicatinglong term persistence of TAC-T cells.

The data illustrates that TAC-T cells destroy tumor cells likely via amechanism that mimics the natural process of T cell activation. The TACtechnology illustrates 1) strong efficacy in liquid, 2) in vivoproliferation, 3) T cell persistence, protecting mice from re-challenge,and 4) cell expansion following T cell administration.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A BCMA (B Cell Maturation Antigen) T cell-antigencoupler (BCMA-TAC) polypeptide, comprising the amino acid sequence setforth in SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO: 62.2. The BCMA-TAC polypeptide of claim 1, wherein the amino acid sequenceof SEQ ID NO: 56 is encoded by the nucleic acid sequence set forth inSEQ ID NO: 55, the amino acid sequence of SEQ ID NO: 58 is encoded bythe nucleic acid sequence set forth in SEQ ID NO: 57, the amino acidsequence of SEQ ID NO: 60 is encoded by the nucleic acid sequence setforth in SEQ ID NO: 59, and the amino acid sequence of SEQ ID NO: 62 isencoded by the nucleic acid sequence set forth in SEQ ID NO:
 61. 3. Anengineered T cell, comprising a recombinant expression vector comprisingthe nucleic acid sequence of SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, or SEQ ID NO: 61, wherein the expression vector encodes a BCMA (BCell Maturation Antigen) T cell-antigen coupler (BCMA-TAC) polypeptideand the BCMA-TAC polypeptide is expressed on the surface of the cell. 4.A pharmaceutical composition, comprising: (a) the T cell of claim 3; and(b) an excipient.
 5. The pharmaceutical composition of claim 4, whereinthe pharmaceutical composition is in a unit dose form.
 6. Thepharmaceutical composition of claim 4, wherein the pharmaceuticalcomposition comprises about 1×10⁴ T cells per kg body weight to about1×10⁸ T cells per kg body weight.
 7. The pharmaceutical composition ofclaim 4, wherein the pharmaceutical composition comprises about0.5-2×10⁹ T cells.
 8. A recombinant expression vector comprising: (a) anucleic acid sequence comprising the sequence set forth in SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO: 61; and (b) a promoterfunctional in a mammalian cell.
 9. A method for treating aBCMA-expressing cancer in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of the T cell of claim 3, wherein administration of said T cellresults in a reduction in cancer cells in the subject.
 10. The method ofclaim 9, wherein the cancer is a B cell malignancy.
 11. The method ofclaim 9, wherein the cancer is multiple myeloma, B cell lymphoma, acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), orNon-Hodgkins Lymphoma.